XLI Bibliography

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The F1F0 ATP synthase is a reversible molecular motor that employs a rotary catalytic cycle to couple a chemiosmotic membrane potential to the formation/hydrolysis of ATP. The multisubunit enzyme contains two copies of the b subunit that form a homodimer as part of a narrow, peripheral stalk structure that connects the membrane (F0) and soluble (F1) sectors. The three-dimensional structure of the b subunit is unknown making the nature of any interactions or conformational changes within the F1F0 complex difficult to interpret. We have used circular dichroism and analytical ultracentrifugation analyses of a series of N- and C-terminal truncated b proteins to investigate its stability and structure. Thermal denaturation of the b constructs exhibited distinct two-state, cooperative unfolding with Tm values between 30 and 40degreeC. CD spectra for the region comprising residues 53-122 (b53-122) showed theta222/theta208= 0.99, which reduced to 0.92 in the presence of the hydrophobic solvent trifluoroethanol. Thermodynamic parameters for b53-122 (DELTAG, DELTAH and DELTACp) were similar to those reported for several nonideal, coiled-coil proteins. Together these results are most consistent with a noncanonical and unstable parallel coiled-coil at the interface of the b dimer.

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Vascular endothelial (VE) cadherin is an endothelial specific cadherin that plays a major role in remodeling and maturation of vascular vessels. Recently, we presented evidence that the extracellular part of VE cadherin, which consists of five homologous modules, associates as a Ca2+-dependent hexamer in solution (Legrand, P., Bibert, S., Jaquinod, M., Ebel, C., Hewat, E., Vincent, F., Vanbelle, C., Concord, E., Vernet, T., and Gulino, D. (2001) J. Biol. Chem. 276, 3581-3588). In an effort to identify which extracellular modules are involved in the elaboration and stability of this hexameric structure, we expressed various VE cadherin-derived fragments overlapping individual or multiple successive modules as soluble proteins, purified each to homogeneity, and tested their propensity to self-associate. Altogether, the results demonstrate that, as their length increases, VE cadherin recombinant fragments generate increasingly complex self-associating structures; although single module fragments do not oligomerize, some two or three module-containing fragments self-assemble as dimers, and four module-containing fragments associate as hexamers. Our results also suggest that, before elaborating a hexameric structure, molecules of VE cadherin self-assemble as intermediate dimers. A synergy between the extracellular modules of VE cadherin is thus required to build homotypic interactions. Placed in a cellular context, this particular self-association mode may reflect the distinctive biological requirements imposed on VE cadherin at adherens junctions in the vascular endothelium.

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The self-association behavior of the Eph-kinases SAM domain has been studied in phosphate buffer, pH 7.4, containing 0.14 M NaCl using concentration-dependent sedimentation equilibrium experiments. Only weak interactions typical for a monomer-dimer equilibrium up to at least 12 mg/mL were observed. Such concentrated solutions require a consideration of the non-ideality expressed by virial coefficients. A special centrifuge equation was used for the global analysis to estimate equilibrium constants based on the thermodynamic activities of the reactants. When neglecting this, the parameters deviate by about 20%. Association constants for dimerization of the EphB2-SAM domain vary between 163 M-1 at 10 degreeC and 395 M-1 at 32 degreeC, indicating hydrophobic forces are involved in the dimerization process. In solutions of about 12 mg/mL, less than 50% dimers are in solution and higher oligomers can be excluded.

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The effects of laser irradiation on molecular mass and conformation of pure chondroitin sulphate dissolved in phosphate buffered saline (PBS) were investigated using size exclusion chromatography/multi-angle light scattering (SEC/MALS) and sedimentation velocity in the analytical ultracentrifuge . In addition, cartilage pieces immersed in buffer were irradiated with a laser in order to study whether cartilage components may diffuse away from the matrix and into the surrounding aqueous medium as a result of laser treatment. Size exclusion chromatography/multi-angle light scattering and sedimentation velocity measurements showed that (a) laser irradiation decreases the molecular mass of chondroitin sulphate and (b) laser irradiation of cartilage induces diffusion of macromolecules into the medium.

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The SCAN domain is a conserved region of 84 residues found predominantly in zinc finger DNA-binding proteins in vertebrates. The SCAN domain appears to control the association of SCAN domain containing proteins into noncovalent complexes and may be the primary mechanism underlying partner choice in the oligomerization of these transcription factors. Here we have overexpressed, purified, and characterized the isolated SCAN domain (amino acids 37-132) from ZNF174. Both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the ZNF174 SCAN domain forms a homodimer. Circular dichroism shows that the isolated SCAN domain dimer has apprx42% alpha-helix. Thermal denaturation experiments indicate that the SCAN domain undergoes a single reversible unfolding transition with a Tm of over 70 degreeC. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration, consistent with a two-state unfolding transition in which folded dimer is in equilibrium with unfolded monomer. These findings demonstrate that the isolated SCAN domain forms a stable dimer and support a model in which the SCAN domain is capable of mediating the selective dimerization of a large family of vertebrate-specific, zinc finger-containing transcription factors.

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Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 muM Kd. Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

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The association of the bacteriophage T4-encoded AsiA protein with the sigma70 subunit of the Escherichia coli RNA polymerase is one of the principal events governing transcription of the T4 genome. Analytical ultracentrifugation and NMR studies indicate that free AsiA is a symmetric dimer and the dimers can exchange subunits. Using NMR, the mutual recognition sites on AsiA and sigma70 have been elucidated. Residues throughout the N-terminal half of AsiA are involved either directly or indirectly in binding to sigma70 whereas the two highly conserved C-terminal regions of sigma70, denoted 4.1 and 4.2, constitute the entire AsiA binding domain. Peptides corresponding to these regions bind tightly to AsiA individually and simultaneously. Simultaneous binding promotes structural changes in AsiA that mimic interaction with the complete AsiA binding determinant of sigma70. Moreover, the results suggest that a significant rearrangement of the dimer accompanies peptide binding. Thus, both conserved regions 4.1 and 4.2 are intimately involved in recognition of AsiA by sigma70. The interaction of AsiA with 4.1 provides a potential explanation of the differential abilities of DNA and AsiA to bind to free sigma70 and a mechanistic alternative to models of AsiA function that rely on binding to a single site on sigma70.

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Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation , and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.

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The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 ANG. There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules, flexible loops close onto the active site. This closure mimics the conformation of the substrate-bound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a specific role to it, a structural role is possible.

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Lrp is a global regulatory protein in Escherichia coli that activates expression of more than a dozen operons and represses expression of another dozen. For some operons, exogenous leucine reduces the extent of Lrp action, for others it potentiates the effect of Lrp, and for yet other operons it has no effect. In an effort to understand how leucine affects Lrp-mediated expression, we examined Lrp self-association and the effect of leucine on self-association using light scattering, chemical cross-linking, and analytical ultracentrifugation . The following results were obtained. (i) Lrp self-associates to a hexadecamer and octamer with the predominant species being hexadecamer at muM concentrations. (ii) Lrp undergoes a leucine-induced dissociation of hexadecamer to octamer. (iii) A mutant Lrp lacking 11 amino acid residues at the C terminus does not form higher-order oligomers, suggesting that the C terminus is involved in subunit association. (iv) At nM concentrations, Lrp dissociates to a dimer. It is proposed that leucine regulates the equilibrium between Lrp oligomers and thus Lrp occupancy of sites within different operons, leading to diverse regulatory patterns.

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Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg2+ ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg2+. Addition of alpha-casein partially substitutes for Mg2+ in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg2+ ions but not by AMP-PNP or ATPgammaS. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl2. Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl2, at pH 8.0 and 20degreeC, and that the assembly reaction is Mg2+ dependent; the mole fraction of hexamer decreases with decreasing MgCl2 to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl2 concentrations yields two assembly models: dimertautmtetramertautmhexamer and timertautmhexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg2+-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg2+, and stimulator, unfolded protein.

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The S100 calcium-binding proteins are implicated in signal transduction, motility, and cytoskeletal dynamics. The three-dimensional structure of several S100 proteins revealed that the proteins form non-covalent dimers. However, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible equilibrium (Kd = 4 +- 2 muM). The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. Helix IV is known to form the major part of the dimerization interface in homologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix IV, namely Phe-72, Tyr-75, Phe-78, and Leu-79, are essential for dimerization in vivo. A homology model demonstrated that these residues form a hydrophobic cluster on helix IV. Their role is to stabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent data indicating that S100 proteins can form heterodimers.

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The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein-smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein. From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein-osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins.

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Adipocyte complement-related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived hormone that affects lipid and glucose metabolism in muscle and liver, but its physical and biochemical properties are poorly characterized. Here we have used several approaches to show that Acrp30 expressed in and purified from Escherichia coli and human embryonic kidney 293T cells forms trimers and hexamers; 293T cells also produce a higher molecular weight species. Similar Acrp30 oligomers were found in mouse serum as well as in 3T3-L1 adipocyte-conditioned medium, although in different proportions. In parallel, we assessed whether Acrp30 is a signaling molecule by searching for promoter or enhancer elements that respond to Acrp30 or its isolated trimeric globular C-terminal domain, gAcrp30. Acrp3O addition to C2C12 myocytes or myotubes led to activation of NF-kappaB transcription factor in a manner dependent upon phosphorylation and degradation of IkappaB-alpha. Importantly, only hexameric and larger isoforms of Acrp30 activated NF-kappaB; trimeric Acrp30 or gAcrp30 could not activate NF-kappaB. Our data indicate that oligomerization of Acrp30 is important for at least some of its biological activities, and changes in the relative abundance of each oligomeric isoform in plasma may regulate Acrp3O activity.

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Development of specifically labeled chemokines that retain their biological properties should be useful for analyzing their mechanisms of action both under physiological and pathological conditions. Here, we report the chemical synthesis and characterization of RANTES (regulated upon activation normal T cell expressed and secreted) derivatives that were biotinylated at residues 1, 25, 33, 45, or 67. Gel filtration and ultracentrifugation experiments showed that biotinylation at position 45 or 67 decreased the aggregation tendency of the chemokine to a dimeric state. Competition experiments, using a stably transfected CHO-K1 cell line overexpressing human CCR5, a RANTES receptor, indicated that derivatives biotinylated at positions 1, 25, and 67 bound to CCR5 with the same affinity as native RANTES. Flow cytometry analysis showed that RANTES biotinylated at residue 67 (B67-RANTES) bound more efficiently to primary macrophages than the other derivatives. Such binding was dependent on cell surface glycosaminoglycans (GAGs) since it was reduced when macrophages or HeLa cells expressing or not CCR5 were first treated with GAG-specific enzymes. In addition, B67-RANTES modulated CCR5 expression on lymphocytes and elicited chemotaxis of monocytes in the same manner as unmodified RANTES. Thus, B67-RANTES acts as a CCR5 agonist and may be useful to study the role of RANTES in pathologies such as, for example, human immunodeficiency virus (HIV) infection and inflammatory disorders.

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The (+)-pinoresinol-forming dirigent protein is the first protein capable of stereoselectively coupling two coniferyl alcohol derived radical species, in this case to give the 8-8' linked (+)-pinoresinol. Only dimeric cross-linked dirigent protein structures were isolated when 1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide was used as cross-linking agent, whereas the associated oxidase, presumed to generate the corresponding free radical substrate, was not detected. Native Forsythia intermedia dirigent protein isoforms were additionally subjected to MALDI-TOF and ESI-MS analyses, which established the presence of both monomeric masses of 23-25 kDa and dimeric dirigent protein species ranging from 46 to 49 kDa. Analytical ultracentrifugation , sedimentation velocity, and sedimentation equilibrium analyses of the native dirigent protein in open solution confirmed further its dimeric nature as well as a propensity to aggregate, with the latter being dependent upon both temperature and solution ionic strength. Circular dichroism analysis suggested that the dirigent protein was primarily composed of beta-sheet and loop structures.

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The design of large macromolecular assemblies is an endeavor with implications for protein engineering as well as nanotechnology. A hierarchic approach was used to design an antiparallel hexameric, tubular assembly of helices. In previous studies, a domain-swapped, dimeric three-helix bundle was designed from first principles. In the crystal lattice, three dimers associate around a 3-fold rotational axis to form a hexameric assembly. Although this hexameric assembly was not observed in solution, it was possible to stabilize its formation by changing three polar residues per monomer to hydrophobic (two Phe and one Trp) residues. Molecular models based on the crystallographic coordinates of DSD (PDB accession code 1G6U) show that these side-chains pack in the central cavity (the "supercore") of the hexameric bundle. Analytical ultracentrifugation , fluorescence spectroscopy, CD spectroscopy, and guanidine-HCl denaturation were used to determine the assembly of the hexamer. To probe the requirements for stabilizing the hexamer, we systematically varied the polarity and steric bulk of one of the Phe residues in the supercore of the hexamer. Depending on the nature of this side-chain, it is possible to modulate the stability of the hexamer in a predictable manner. This family of hexameric proteins may provide a useful framework for the construction of proteins that change their oligomeric states in response to binding of small molecules.

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Bovine beta-lactoglobulin, a major protein in cow's milk composed of nine beta-strands (betaA-betaI) and one alpha-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular beta-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation . Various I-strand mutants showed decreases in Kalpha, suggesting that the intermolecular beta-sheet is essential for dimer formation. By substituting either Asp33 or Arg40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in Kalpha was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg40 on one monomer and Asp33 on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg33 and Asp40 of different molecules. These results suggested that protein-protein interactions of bovine beta-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.

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Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes.

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A novel inhibitor against maltose-producing alpha-amylase was prepared via stepwise degradation of a high-molecular-weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be alpha-D-glucopyranosyl-alpha-acarviosinyl-D-glucopyranose (GlcAcvGlc) by 13C NMR and MALDI-TOF/MS. Progress curves of PNPG2 hydrolysis by various amylolytic enzymes, including MGase, ThMA, and CDase I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. Analytical ultracentrifugation and X-ray crystallography analyses revealed that the presence of GlcAcvGlc increased the dimerization of ThMA. The formation of dimer complexed with GlcAcvGlc might induce a conformational change in ThMA, leading to a two-step inhibition process. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.

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Refolding of the acid-unfolded precursor to mitochondrial aspartate aminotransferase (pmAAT) is inhibited when cytosolic Hsc70 is included in the refolding reaction (Artigues, A., Iriarte, A., and Martinez-Carrion, M. (1997) J. Biol. Chem. 272, 16852-16861). At low molar excess of Hsc70 pmAAT is recovered in insoluble aggregates containing equal amounts of Hsc70. However, in the presence of a large excess of Hsc70, refolding of pmAAT is still arrested, but the enzyme remains in solution. Similar behavior was observed with two other cytosolic chaperones, bovine Hsp90 and yeast Ydj1. Co-immunoprecipitation of pmAAT using Hsc70 antibodies confirmed the formation of soluble Hsc70-pmAAT complexes at high concentrations of the chaperone. Data from analytical centrifugation, sedimentation in glycerol gradients, and partial purification of the soluble complexes indicate that multiple Hsc70 molecules bind per pmAAT polypeptide chain. The absence of catalytic activity together with the protease susceptibility of pmAAT bound to Hsc70, Hsp90, or Ydj1 suggest that these chaperones bind and maintain pmAAT in a partially unfolded state, analogous to the import-competent conformation of the protein synthesized in cell-free extracts. Remarkably, the purified pmAAT bound to Hsc70 or Ydj1, but not to Hsp90, is imported by isolated mitochondria in a reticulocyte lysate-dependent manner. Thus, both Hsc70 and Ydj1 can trap an import-competent folding intermediate of pmAAT, but productive binding and import into mitochondria require the collaboration of additional cytosolic factors from the lysate.

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MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody VH and VL domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact VH-VL-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9 X 103-2.2 X 103 M-1. Sedimentation velocity experiments using the time derivative g(s*) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by 1H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degreeC, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the VH and VL domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for seven other scFv molecules have shown that, while the contact residues for symmetric back-to-back dimer formation in MFE-23 are not fully conserved, in principle, back-to-back contacts can be formed in these too. This offers possibilities for the creation of other forms of scFv molecules.

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Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030-1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1-P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1-P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E. coli equivalent RNA domain, were tested for their function by replacing with the E. coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1-P2 heterodimer is required for subsequent formation of the P0(P1-P2)2 complex and its functional rRNA binding in silkworm ribosomes.

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Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 ANG resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 ANG resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 ANG. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg2+ in vivo. In the free structure, a Mg2+ is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

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Mac-2-binding protein (M2BP) is a secreted glycoprotein suggested to have a role in host defense. It forms linear and ring-shaped oligomers, with each ring segment being composed of two monomers. We have produced recombinant human M2BP fragments comprising domains 1 and 2 (M2BP-1,2) and domains 3 and 4 (M2BP-3,4) in 293 human kidney cells to characterize structural and functional properties of M2BP. Both fragments were obtained in a native and glycosylated form, as analyzed by CD spectroscopy, trypsin susceptibility, and enzymatic deglycosylation. These results strongly suggest that both fragments are autonomous folding units. All three potential N-glycosylation sites in M2BP-1,2 and all four in M2BP-3,4 were found to be occupied. M2BP-1,2 expressed in tunicamycin-treated cells contained no glycosyl residues, indicating that O-glycosylation is not occurring. Ultracentrifugation revealed that M2BP-1,2 is homogeneously dimeric in the nanomolar range reflecting the properties of intact M2BP. Domain 2 (BTB/POZ domain) is thus identified as the dimerization domain of M2BP, because it has been formerly shown that recombinant domain 1 is monomeric. M2BP-3,4 showed a concentration-dependent self-association, and aggregates of different size and shape were shown by electron microscopy. In contrast to this irregular aggregation of M2BP-3,4, it has been formerly shown that a fragment comprising domains 2-4 still has the ability to form ring-like structures, although the rings are protein-filled, and thus domain 2 appears to be indispensable for ring formation. Solid phase assays showed that M2BP-3,4 contains binding sites for galectin-3, nidogen, and collagens V and VI, whereas M2BP-1,2 is inactive in binding. Both fragments showed no cell adhesive activity in contrast to native M2BP, suggesting that a concerted binding action and/or multivalent interactions of rings are necessary for cell attachment.

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The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36Mut(e,g) and N36Mut(a,d), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36Mut(e,g) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36Mut(a,d) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36Mut(a,d) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36Mut(e,g) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC50 apprx 308 nM versus apprx 16 muM). These results indicate that N36Mut(e,g) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36Mut(e,g) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36Mut(e,g) with the pre-hairpin intermediate is presented.

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The clinical effectiveness of succinylated gelatin as a plasma substitute depends strongly on its molar mass, determined conventionally by size exclusion chromatography (SEC). This study evaluates different SEC calibration standards in comparison with two independent "absolute" methods for determining the weight average molar mass (Mw) of a succinylated gelatin sample. SEC calibrated using succinylated gelatin fractions correlated well with size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) and sedimentation equilibrium whereas SEC calibrated with unmodified gelatin, sodium polystyrene sulfonates or pullulans overestimated Mw by over 20%. Universal calibration was equivocal. The problems associated with the preparation of succinylated gelatin fractions suggest that an absolute method such as SEC-MALLS may be a more suitable choice for determining the Mw in succinylated gelatins.

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Matrilins constitute a family of four oligomeric extracellular proteins that are involved in the development and homeostasis of cartilage and bone. To reveal their homo- and heterotypic oligomerization propensities, we analyzed the four human matrilin coiled-coil domains by biochemical and biophysical methods. These studies not only confirmed the homo- and heterotypic oligomerization states reported for the full-length proteins but revealed seven novel matrilin isoforms. Specific heterotrimeric interactions of variable chain stoichiometries were observed between matrilin-1 and matrilin-2, matrilin-1 and matrilin-4, and matrilin-2 and matrilin-4. In addition, matrilin-1 formed two different specific heterotetramers with matrilin-3. Interestingly, a distinct heterotrimer consisting of three different chains was formed between matrilin-1, matrilin-2, and matrilin-4. No interactions, however, were observed between matrilin-2 and matrilin-3 or between matrilin-3 and matrilin-4. Both homo- and heterotypic oligomers folded into parallel disulfide-linked structures, although coiled-coil formation was not dependent on disulfide bridge formation. Our results indicate that the heterotypic preferences seen for the matrilin coiled-coil domains are the result of the packing of the hydrophobic core rather than ionic interactions. Mass spectrometry revealed that the concentrations of the individual chains statistically determined the stoichiometry of the heteromers, suggesting that formation of the different matrillin chain combinations is controlled by expression levels.

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Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and RNA polymerase holoenzyme associate to form a 2:2 complex in vitro. No complexes of lower stoichiometry (1:1, 2:1, 1:2) were detected over a wide range of CAP and RNA polymerase concentrations, suggesting that the interaction is highly cooperative. The absence of higher stoichiometry complexes, even in the limit of high (protein), suggests that the 2:2 species represents binding saturation for this system. The 2:2 pattern of complex formation is robust. A lower-limit estimate of the formation constant in our standard buffer (40 mM Tris (pH 7.9), 10 mM MgCl2, 0.1 mM dithiothreitol, 5% glycerol, 100 mM KCl) is 2 X 1020 M-3. The qualitative pattern of association is unchanged over the temperature range 4 degreeC ltoreq T ltoreq 20 degreeC, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 muM cAMP to the reaction mix. These results limit the possible mechanisms of CAP-polymerase association. In addition, they support the idea that CAP binding may influence the availability of the monomeric form of RNA polymerase that mediates transcription at many promoters.

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The b subunit of E. coli F0F1-ATPase links the peripheral F1 subunits to the membrane-integral F0 portion and functions as a "stator", preventing rotation of F1. The b subunit is present as a dimer in ATP synthase, and residues 62-122 are required to mediate dimerization. To understand how the b subunit dimer is formed, we have studied the structure of the isolated dimerization domain, b62-122. Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) indicate that the b62-122 dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 ANG, and a radius of gyration of 27 ANG, values that are consistent with an alpha-helical coiled-coil structure. The crystal structure of b62-122 has been solved and refined to 1.55 ANG. The protein crystallized as an isolated, monomeric alpha helix with a length of 90 ANG. Combining the crystal structure of monomeric b62-122 with SAXS data from the dimer in solution, we have constructed a model for the b62-122 dimer in which the two helices form a coiled coil with a right-handed superhelical twist. Analysis of b sequences from E. coli and other prokaryotes indicates conservation of an undecad repeat, which is characteristic of a right-handed coiled coil and consistent with our structural model. Mutation of residue Arg-83, which interrupts the undecad pattern, to alanine markedly stabilized the dimer, as expected for the proposed two-stranded, right-handed coiled-coil structure.

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The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn afwdarwa' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.

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Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.

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alpha-Helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation . We show that all peptides are highly alpha-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.

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Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain-binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo.

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Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. 15N and 1H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.

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Given the importance of protein complexes as therapeutic targets, it is necessary to understand the physical chemistry of these interactions under the crowded conditions that exist in cells. We have used sedimentation equilibrium to quantify the enhancement of the reversible homodimerization of alpha-chymotrypsin by high concentrations of the osmolytes glucose, sucrose, and raffinose. In an attempt to rationalize the osmolyte-mediated stabilization of the alpha-chymotrypsin homodimer, we have used models based on binding interactions (transfer-free energy analysis) and steric interactions (excluded volume theory) to predict the stabilization. Although transfer-free energy analysis predicts reasonably well the relatively small stabilization observed for complex formation between cytochrome c and cytochrome c peroxidase, as well as that between bobtail quail lysozyme and a monoclonal Fab fragment, it underestimates the sugar-mediated stabilization of the alpha-chymotrypsin dimer. Although predictions based on excluded volume theory overestimate the stabilization, it would seem that a major determinant in the observed stabilization of the alpha-chymotrypsin homodimer is the thermodynamic nonideality arising from molecular crowding by the three small sugars.

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Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation , we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EIIMtl. For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EIIMtl and four other membrane (transport) proteins could be deduced within 10% error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.

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Nicotinamide/nicotinate mononucleotide (NMN/NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD+ and NADP+. Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH2-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions.

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The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-ANG resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.

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Bovine heart cytochrome c oxidase (CcO), solubilized by either nonionic detergents or phospholipids, completely dimerizes upon the addition of bile salts, e.g., sodium cholate, sodium deoxycholate, or CHAPS. Bile salt induced dimerization occurs whether dodecyl maltoside, decyl maltoside, or Triton X-100 is the primary solubilizing detergent or the enzyme is dispersed in phosphatidylcholine, phosphatidylethanolamine, or mixtures thereof. In each case, complete CcO dimerization can be verified by sedimentation velocity and sedimentation equilibrium after correction for bound detergent and/or phospholipid. The relative concentration of the bile salt is critical for production of homogeneous, dimeric CcO. For example, enzyme solubilized by 2 mM detergent requires an equal molar concentration of sodium cholate. Similarly, enzyme dispersed in 20 mM phospholipid requires 50 mM sodium cholate, concentrations that are commonly used to reconstitute CcO into small unilamellar vesicles. Bile salts do more than just stabilize dimeric CcO and prevent detergent-induced dissociation into monomers. They are able to completely reverse detergent-induced monomerization and cause completely monomeric CcO to reassociate. Dimeric CcO so generated is no more stable than the original complex and easily dissociates into monomers if the bile salt is removed. The dimerization process is dependent upon a full complement of subunits; e.g., if subunits VIa and VIb are removed, the resulting monomeric CcO will not reassociate upon the addition of sodium cholate. These results support four important consequences: (1) dissociation of dimeric CcO into monomers is reversible; (2) stable dimers can be produced under solution conditions; (3) dimers can be stabilized even at relatively high pH and low enzyme concentration; and (4) subunits VIa and VIb are required for dimerization.

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In homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM), cysteine 14 of each the two subunits forms part of the dimer interface. This residue is central for the catalysis and stability of TbTIM. Cys14 was changed to the other 19 amino acids to determine the characteristics that the residue must have to yield catalytically competent stable enzymes. C14A, C14S, C14P, C14T, and C14V TbTIMs were essentially wild type in activity and stability. Mutants with Asn, Arg, and Gly had low activities and stabilities. The other mutants had less than 1% of the activity of TbTIM. One of the latter enzymes (C14F) was purified to homogeneity. Size exclusion chromatography and equilibrium sedimentation studies showed that C14F TbTIM is a monomer, with a kcat apprx1000 times lower and a Km apprx6 times higher than those of TbTIM. In C14F TbTIM, the ratio of the elimination (methylglyoxal and phosphate formation) to isomerization reactions was higher than in TbTIM. Its secondary structure was very similar to that of TbTIM; however, the quantum yield of its aromatic residues was lower. The analysis of the data with the 19 mutants showed that to yield enzymes similar to the wild type, the residue must have low polarity and a van der Waals volume between 65 and 110 ANG3. The results with C14F TbTIM illustrate that the secondary structure of TbTIM can be formed in the absence of intersubunit contacts, and that it has sufficient tertiary structure to support catalysis.

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The K homology module, one of the most common RNA-binding motifs, is present in multiple copies in both prokaryotic and eukaryotic regulatory proteins. Increasing evidence suggests that self-aggregation of KH modules has a functional role. We have used a combination of techniques to characterize the behavior in solution of the third KH domain of Nova-1, a paradigmatic KH protein. The possibility of working on the isolated module allowed us to observe specifically the homodimerization and RNA-binding properties of KH domains. We provide conclusive evidence that self-association of Nova-1 KH3 occurs in solution even in the absence of RNA. Homodimerization involves a specific protein/protein interface. We also studied the dynamical behavior of Nova-1 KH3 in isolation and in complex with RNA. These data provide a model for the mechanism of KH/RNA recognition and suggest functional implications of dimerization in KH complexes. We discuss our findings in the context of the whole KH family and suggest a generalized mode of interaction.

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The role of amino acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study alpha to beta conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an alpha-helical leucine zipper and a beta-hairpin. The parent peptide, Template-alpha, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation . To introduce beta-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-alphaT. Unlike the parent peptide, Template-alphaT underwent a heat-inducible switch to beta-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilization of the native states facilitate transitions to amyloid, destabilization of the leucine-zipper form of Template-alphaT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative beta-hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template-alpha, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favor beta-structure promote amyloid formation.

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SecA, a homodimeric protein involved in protein export in Escherichia coli, exists in the cell both associated with the membrane translocation apparatus and free in the cytosol. SecA is a multifunctional protein involved in protein localization and regulation of its own expression. To carry out these functions, SecA interacts with a variety of proteins, phospholipids, nucleotides, and nucleic acid and shows two enzymic activities. It is an ATPase and a helicase. Its role during protein localization involves interaction with the precursor polypeptides to be exported, the cytosolic chaperone SecB, and the SecY subunit of the membrane-associated translocase, as well as with acidic phospholipids. At the membrane, SecA undergoes a cycle of binding and hydrolysis of ATP coupled to conformational changes that result in translocation of precursors through the cytoplasmic membrane. The helicase activity of SecA and its affinity for its mRNA are involved in regulation of its own expression. SecA has been reported to exist in at least two conformational states during its functional cycle. Here we have used analytical centrifugation, as well as column chromatography coupled with multiangle light scatter, to show that in solution SecA undergoes at least two monomer-dimer equilibrium reactions that are sensitive to temperature and to concentration of salt.

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The light-inducible carB operon encodes all but one of the structural genes for carotenogenesis in Myxococcus xanthus. It is transcriptionally controlled by two proteins expressed from two unlinked genetic loci: CarS from the light-inducible carQRS operon, and CarA from the light-independent carA operon. CarA represses transcription from the carB promoter (PB) in the dark, and CarS counteracts this on illumination. The CarA sequence revealed a helix-turn-helix DNA-binding motif of the type found in bacterial MerR transcriptional factors, whereas CarS contains no known DNA-binding motif. Here, we examine the molecular interplay between CarA and CarS. We demonstrate the following. (i) Whereas CarS exhibits no DNA binding in vitro, CarA binds specifically to a region encompassing PB to form at least two distinct complexes. (ii) A palindrome located between positions -46 and -63 relative to the transcription start point is essential but not sufficient for the formation of the two CarA-DNA complexes observed. (iii) CarS abrogates the specific DNA binding of CarA. CarA is therefore a repressor and CarS an antirepressor. (iv) CarS physically interacts with CarA; thus, the functional interaction between them is mediated by protein-protein interactions.

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The subunit interface of the homodimeric hemoglobin from Scapharca inaequivalvis, HbI, is stabilized by a network of interactions that involve several hydrogen-bonded structural water molecules, a hydrophobic patch, and a single, symmetrical salt bridge between residues Lys-30 and Asp-89. Upon mutation of Lys-30 to Asp, the interface is destabilized markedly. Sedimentation equilibrium and velocity experiments allowed the estimate of the dimerization constants for the unliganded (K1,2 D = 8 X 104 M-1) and for the CO-bound (K1,2 L = 1 X 103 M-1) and oxygenated (K1,2 L = 70 M-1) derivatives. For the oxygenated derivative, the destabilization of the subunit interface with respect to native HbI corresponds to about 8 kcal/mol, an unexpectedly high figure. In the K30D mutant, at variance with the native protein, oxygen affinity and cooperativity are strongly dependent on protein concentration. At low protein concentrations (e.g. 1.2 X 10-5 M heme), at which the monomeric species becomes significant also in the unliganded derivative, oxygen affinity increases and cooperativity decreases. At protein concentrations where both derivatives are dimeric (e.g. 3.3 X 10-3 M heme), both cooperativity and oxygen affinity decrease. Taken together, the experimental data indicate that in the K30D mutant, the mechanism of cooperativity is drastically altered and is driven by a ligand-linked monomer-dimer equilibrium rather than being based on a direct heme-heme communication as in native HbI.

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The response regulator SpoOA is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated SpoOA is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of SpoOA function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that SpoOA forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of SpoOA, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of SpoOA.

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Our basic understanding of how to combat fungal infections has not kept pace with the recent sharp rise in life-threatening cases found particularly among immuno-compromised individuals. Current investigations for new potential antifungal agents have focused on antimicrobial peptides, which are used as a cell-free defense mechanism in all organisms. Unfortunately, despite their high antibacterial activity, most of them are not active toward fungi, the reason of which is not clear. Here, we present a new approach to modify an antibacterial peptide, a magainin analogue, to display antifungal activity by its conjugation with lipophilic acids. This approach has the advantage of producing well-defined changes in hydrophobicity, secondary structure, and self-association. These modifications were characterized in solution at physiological concentrations using CD spectroscopy, tryptophan fluorescence, and analytical ultra-centrifugation. In order of increasing hydrophobicity, the attachment to the magainin-2 analogue of (i) heptanoic acid results in a monomeric, unordered structure, (ii) undecanoic acid yields concentration-dependent oligomers of alpha helices, and (iii) palmitic acid yields concentration-independent alpha-helical monomers, a novel lipopeptide structure, which is resistant to proteolytic digestion. Membrane-lipopeptide interactions and the membrane-bound structures were studied using fluorescence and ATR-FTIR in PC/PE/PI/ergosterol (5/2.5/2.5/1, w/w) SUV, which constitute the major components of Candida albicans bilayers. A direct correlation was found between oligomerization of the lipopeptides in solution and potent antifungal activity. These results provide insight to a new approach of modulating hydrophobicity and self-assembly of antimicrobial peptides in solution, without altering the sequence of the peptidic chain. These studies also provide a general means of developing a new group of lipopeptide candidates as therapeutic agents against fungal infections.

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The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degreeC and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 M urea. The enzyme activity was activated by urea up to 1 M but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mM Mn2+, the urea denaturation pattern of malic enzyme changed to monophasic. Mn2+ helped the enzyme to resist phase I urea denaturation. The (urea)0.5 for the enzyme inactivation shifted from 2.2 to 3.8 M. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 M urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 M. Mn2+ retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn2+. These results indicate that the Mn2+ has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.

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Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation , and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a KD value of 63 muM3 (where KD = (monomer)4/(tetramer)) so that at moderate concentrations (10 muM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm X 9.0 nm X 6.5 nm, that appears to contain a central pore.

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The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation , fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degreeC and unfolding enthalpy (DELTAHcal) of 800 kcalcntdot(mol of trimer)-1. Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degreeC), unfolding enthalpy (from 800 to 260 kcalcntdot(mol of trimer)-1), and DELTAHcal/DELTAHvH ratio (from 3.0 to apprx1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.

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The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg2+-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20degreeC. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants (apprx2X106 (M monomer)-1) compared to those of dephospho-EI and EI(H189A) at 20degreeC. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl2 (KA' gtoreq108 M-1 at 4 or 20degreeC), as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg2+ also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (Tm) for unfolding the C-terminal domain by apprx18degreeC and that for the N-terminal domain by apprx9degreeC to Tmaxsimeq63degreeC, giving a value of KD'simeq3 muM PEP at 45degreeC. PEP alone also promotes the dimerization of EI(H189A) but only increases Tm apprx5 degreeC for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of KD'simeq0.2 mM for PEP dissociation in the absence of Mg2+ at 45degreeC. In contrast, the dimerization constant of phospho-EI at 20degreeC is the same in the absence and presence of 5 mM PEP and 2 mM MgCl2. Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg2+ are important determinants of both the conformational stability and dimerization of dephospho-EI.

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Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait. Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins. Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif. Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high alpha-helical content and moderate stability. NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases. Unlike previously characterized coiled-coils, backbone 15N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region. Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range. NMR analyses suggest a rotationally symmetric trimer. Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.

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The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into beta-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2-HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum.

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NMR spectra of human lymphotactin (hLtn), obtained under various solution conditions, have revealed that the protein undergoes a major conformational rearrangement dependent on temperature and salt concentration. At high salt (200 mM NaCl) and low temperature (10degreeC), hLtn adopts a chemokine-like fold, which consists of a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix (Kuloglu, E. S., McCaslin, D. R., Kitabwalla, M., Pauza, C. D., Markley, J. L., and Volkman, B. F. (2001) Biochemistry 40, 12486-12496). We have used NMR spectroscopy, sedimentation equilibrium, and intrinsic fluorescence to monitor the reversible conformational change undergone by hLtn as a function of temperature and ionic strength. We have used two-, three- and four-dimensional NMR spectroscopy of isotopically enriched protein samples to determine structural properties of the conformational state stabilized at 45degreeC and 0 mM NaCl. Patterns of NOEs and 1Halpha and 13C chemical shifts show that hLtn rearranges under these conditions to form a four-stranded, antiparallel beta-sheet with a pattern of hydrogen bonding that is completely different from that of the chemokine fold stabilized at 10degreeC and 200 mM NaCl. The C-terminal alpha-helix observed at 10degreeC and 200 mM NaCl, which is conserved in other chemokines, is absent at 45degreeC and no salt, and the last 38 residues of the protein are completely disordered, as indicated by heteronuclear 15N-1H NOEs. Temperature dependence of the tryptophan fluorescence of hLtn in low and high salt confirmed that the chemokine conformation is stabilized by increased ionic strength. Sedimentation equilibrium analytical ultracentrifugation showed that hLtn at 40degreeC in the presence of 100 mM NaCl exists mainly as a dimer. Under near physiological conditions of temperature, pH, and ionic strength, both the chemokine-like and non-chemokine-like conformations of hLtn are significantly populated. The functional relevance of this structural interconversion remains to be elucidated.

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With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degreeC or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degreeC, respectively, compared with 98.3 and 98.5 degreeC for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (Km(NADP) of 55.2 and 44.4 muM, respectively) whereas PfIDH was NAD-dependent (Km(NAD) of 68.3 muM). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.

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Human and simian immunodeficiency viruses infect host lymphoid cells by binding CD4 molecules via their gp160 envelope glycoproteins. Biochemical studies on recombinant SIVmac32H (pJ5) envelope ectodomain gp140 precursor protein show that the envelope is a trimer. Using size exclusion chromatography, quantitative amino acid analysis, analytical ultracentrifugation , and CD4-based competition assay, we demonstrate that the stoichiometry of CD4 receptor-oligomeric envelope interaction is 1:1. By contrast, Fab fragments of both neutralizing and non-neutralizing monoclonal antibodies bind at a 3:1 ratio. Thus, despite displaying equivalent CD4 binding sites on each of the three gp140 protomers within an uncleaved trimer, only one site binds the soluble 4-domain human CD4 extracellular segment. The anti-cooperativity and the faster koff of gp140 trimer:CD4 versus gp120 monomer:CD4 interaction suggest that CD4-induced conformational change is impeded in the intact envelope. The implications of these findings for immunity against human immunodeficiency virus and simian immunodeficiency virus are discussed.

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H2A.Z and H2A.1 nucleosome core particles and oligonucleosome arrays were obtained using recombinant versions of these histones and a native histone H2B/H3/H4 complement reconstituted onto appropriate DNA templates. Analysis of the reconstituted nucleosome core particles using native polyacrylamide gel electrophoresis and DNase I footprinting showed that H2A.Z nucleosome core particles were almost structurally indistinguishable from its H2A.1 or native chicken erythrocyte counterparts. While this result is in good agreement with the recently published crystallographic structure of the H2A.Z nucleosome core particle (Suto, R. K., Clarkson, M J., Tremethick, D. J., and Luger, K. (2000) Nat. Struct. Biol. 7, 1121-1124), the ionic strength dependence of the sedimentation coefficient of these particles exhibits a substantial destabilization, which is most likely the result of the histone H2A.Z-H2B dimer binding less tightly to the nucleosome. Analytical ultracentrifuge analysis of the H2A.Z 208-12, a DNA template consisting of 12 tandem repeats of a 208-base pair sequence derived from the sea urchin Lytechinus variegatus 5 S rRNA gene, reconstituted oligonucleosome complexes in the absence of histone H1 shows that their NaCl-dependent folding ability is significantly reduced. These results support the notion that the histone H2A.Z variant may play a chromatin-destabilizing role, which may be important for transcriptional activation.

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The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP4). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultra-centrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP4 sites have roughly equivalent affinities with dissociation constants of about 4 muM. Mutational analysis of the FP4 motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.

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Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation , and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

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Efforts to understand the molecular basis of human immunodeficiency virus (HIV) envelope glycoprotein function have been hampered by the inability to generate sufficient quantities of homogeneous material. We now report on the high level expression, purification, and characterization of soluble HIV gp140 ectodomain proteins in Chinese hamster ovary-Lec3.2.8.1 cells. Gel filtration and analytical ultracentrifugation show that the uncleaved ADA strain-derived gp140 proteins are trimeric without further modification required to maintain oligomers. These spike proteins are native as judged by soluble CD4 (sCD4) (KD = 1-2 nM) and monoclonal antibody binding studies using surface plasmon resonance. CD4 ligation induces conformational change in the trimer, exposing the chemokine receptor binding site as assessed by 17b monoclonal antibody reactivity. Lack of anti-cooperativity in sCD4-ADA trimer interaction distinct from that observed with sCD4-SIV mac32H implies quaternary structural differences in ground states of their respective spike proteins.

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Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe2+) form, indicating a His-Fe2+-His binding scheme. O2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe3+) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (kon) and a slow dissociation rate (koff) for both O2 and CO, indicating a high intrinsic affinity for these ligands. However, because the rate-limiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O2 and CO binding is suggested to be slow in vivo. Because of this competition, the observed O2 affinity of recombinant human neuroglobin is average (1 torr at 37 degreeC). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37 degreeC by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E'o = -129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfide-dependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.

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Calpain is a heterodimeric Ca2+-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca2+ on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca2+ and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca2+ concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca2+, but this was fully reversible by EDTA. With Ca2+ in 0.2 M NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca2+ gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca2+ was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca2+-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.

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A method is described whereby sedimentation velocity is combined with equilibrium dialysis to determine the net charge (valence) of a protein by using chromate as an indicator ion for assessing the extent of the Donnan redistribution of small ions. The procedure has been used in experiments on bovine serum albumin under slightly alkaline conditions (pH 8.0, I 0.05) to illustrate its application to a system in which the indicator ion and protein both bear net negative charge and on lysozyme under slightly acidic conditions (pH 5.0, I 0.10) to illustrate the situation where chromate is a counterion.

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Early endosome autoantigen localization to early endosomes is mediated by a C-terminal region, which includes a calmodulin binding motif, a Rab5 interaction site, and a FYVE domain that selectively binds phosphatidyl inositol 3-phosphate. The crystal structure of the C-terminal region bound to inositol 1,3-bisphosphate reveals an organized, quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal localization in which domain organization, dimerization, and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. A unique mode of membrane engagement deduced from the quaternary structure of the C-terminal region provides insight into the structural basis of endosome tethering.

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We have isolated from a human prostate cDNA library a cDNA encoding a novel member of the S100 family of EF-hand proteins. The encoded 99-amino acid protein, designated S100Z, is capable of interacting with another member of the family, S100P. S100Z cDNA was cloned into a bacterial expression system, and the S100Z protein was purified to homogeneity from bacterial lysates by a combination of hydrophobic column and gel-filtration chromatography. Direct amino acid sequencing of the 20 N-terminal amino acids confirmed that the sequence of the recombinant protein is identical to the sequence deduced from the cDNA. Low-resolution structural data have been obtained using circular dichroism and fluorescence spectroscopies, and equilibrium analytical centrifugation. These results show that S100Z is a dimeric, predominantly alpha-helical protein. Addition of calcium to a solution of S100Z changes the fluorescence intensity of the protein, indicating that S100Z is capable of binding calcium ions. Analysis of the calcium-binding isotherm indicates the existence of two calcium-binding sites with apparent affinities on the order of 5X106 and 102 M-1. Binding of calcium results in conformational changes and exposure of hydrophobic surfaces on the protein. Using a PCR-based assay, we have detected differences in the expression level of S100Z mRNA in various tissues. The highest levels were found in spleen and leukocytes. S100Z gene expression appears to be deregulated in some tumor tissues, compared to expression in their normal counterparts.

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In principle, equilibrium analytical ultracentrifugation (AU) can be used to quantify the binding stoichiometry and affinity between small-molecule ligands and proteins in aqueous solution. We show here that heteromeric binding constants can be determined using a data-fitting procedure which utilizes a postfitting computation of the total amount of each component in the centrifuge cell. The method avoids overconstraining the fitting of the radial concentration profiles, but still permits unique binding constants to be determined using measurements at a single wavelength. The computational program is demonstrated by applying it to data obtained with mixtures of a 500-Da molecule and interleukin-2, a 16-kDa protein. The 1:1 binding stoichiometry and heteromeric dissociation constants (Kab) determined from centrifuge data at two different wavelengths are within 4-9 muM range independently determined from a functional assay. Values for Kab have been obtained for ligands with affinities as weak as 500 muM. This AU method is applicable to compounds with significant UV absorbance (apprx0.2) at concentrations within apprx5- to 10-fold of their Kab. The method, which has been incorporated into a user procedure for Igor-Pro (Wavemetrics, Oswego, OR), is included as supplementary material.

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The relation between the quaternary structure and the substrate specificityof Thermus maltogenic amylase (ThMA) has been investigated. Sedimentation diffusion equilibrium ultracentrifugation and gel filtration analyses, in combination with the crystal structure determined recently, have demonstrated that ThMA existed in a monomer/dimer equilibrium. The truncation of ThMA by removing the N-terminal domain, which is composed of 124 amino acid residues, resulted in the complete monomerization of the enzyme (ThMADELTA124) accompanied by a drastic decrease in the activity for beta-cyclodextrin (beta-CD) and a relatively smaller reduction of the activity for starch. Despite the overall low activity of ThMADELTA124, the activity was higher toward starch than beta-CD, and the ratio of the specific activities toward these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KC1 to wild-type ThMA shifted the monomer/dimer equilibrium toward the monomer. In the presence of 1.0 M KCl, the relative activity of ThMA toward beta-CD decreased to 74%, while that for soluble starch increased to 194% compared to the activities in the absence of KCl. Thus, the ThMA monomer and dimer are both inferred to be enzymatically active but with a somewhat different substrate preference. Kinetic parameters of the wild-type and truncated enzymes also are in accordance with the changes in their specific activities. We thus provide evidence in support of a model, which shows that the relative multisubstrate specificity of ThMA is influenced by the monomer/dimer equilibrium of the enzyme.

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Translin is a recently identified nucleic acid binding protein that appears to be involved in the recognition of conserved sequences found at many chromosomal breakpoints. Previous reports indicate that, based on gel filtration analysis and electron microscopy of protein-DNA complexes, translin forms an octameric structure that binds the DNA. In this study, we further examine the possibility of self-association of translin and its interactions with DNA by analytical ultracentrifugation . Sedimentation velocity analysis of translin indicates that the predominant species sediments with a sedimentation coefficient of 8.5 S and has a frictional ratio, f/fo, of 1.35; these data are consistent with the presence of an octamer with an ellipsoidal configuration; a small amount of a component with significantly higher mass is also present. Equilibrium sedimentation studies of translin at three different protein concentrations also indicate that the predominant species present is an octamer with a minor fraction of aggregated species. Neither monomer nor dimer was detected. Sedimentation equilibrium studies of translin with an FITC-labeled single-stranded oligonucleotide were performed to examine the interaction. A novel analysis method has been developed to analyze protein-nucleic acid interactions based on global fitting of scans of 280 and 490 nm to appropriate mathematical models. Utilizing this method, it was determined that the DNA binding species of translin is an octamer binding a single-stranded oligonucleotide with a DELTAGdegree value of -9.49+-0.12 kcal/mol, corresponding to a dissociation constant, Kd, of 84+-17 nM. On the basis of this evidence and electron microscopy, it is envisioned that translin forms an annular structure of eight subunits, hydrodynamically an oblate ellipsoid, which binds DNA at chromosomal breakpoints.

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Myelopoietins (MPOs) are a family of recombinant chimeric proteins that are both interleukin-3 (IL-3) receptor and granulocyte colony-stimulating factor (G-CSF) receptor agonists. In this study, MPO molecules containing one of three different IL-3 receptor agonists linked with a common G-CSF receptor agonist have been examined for their IL-3 receptor binding characteristics. Binding to the alpha-subunit of the IL-3 receptor revealed that the affinity of the MPO molecules was 1.7-3.4-fold less potent than those of their individual cognate IL-3 receptor agonists. The affinity decrease was reflected in the MPO chimeras having approximately 2-fold slower dissociation rates and 2.7-5.5-fold slower association rates than the corresponding specific IL-3 receptor agonists alone. The affinity of binding of the MPO molecules to the heteromultimeric alphabeta IL-3 receptor expressed on TF-1 cells was either 3-, 10-, or 42-fold less potent than that of the individual cognate IL-3 receptor agonist. Biophysical data from nuclear magnetic resonance, near-UV circular dichroism, dynamic light scattering, analytical ultracentrifugation , and size exclusion chromatography experiments determined that there were significant tertiary structural differences between the MPO molecules. These structural differences suggested that the IL-3 and G-CSF receptor agonist domains within the MPO chimera may perturb one another to varying degrees. Thus, the differential modulation of affinity observed in IL-3 receptor binding may be a direct result of the magnitude of these interdomain interactions.

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The synechococcal metallothionein locus smt consists of two divergent genes: smtA coding for the metallothionein SmtA, and smtB coding for the trans-acting regulator SmtB. The latter binds at two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between the two genes. We have determined the binding stoichiometries to the entire operator/promoter DNA and to the separate S1/S2 and S3/S4 half-operator oligonucletides using sedimentation equilibrium and sedimentation velocity measurements. The full promoter/operator DNA binds two SmtB dimers. The hydrodynamic behavior of this complex supports a compact nucleoprotein structure. Each separate S1/S2 and S3/S4 operator sequence also binds two dimers. An equal molar mixture of separate S1/S2 and S3/S4 operator sequences, in excess SmtB, forms a S1/S2-SmtB:SmtB-S3/S4 bridge complex. Combining these results with previously published binding interference data, which showed consecutive S1/S2 and S3/S4 SmtB occupancy on the operator/promoter DNA, we have developed a model for the establishment of the repression complex that appears to involve significant DNA compaction, presumably DNA bending, stabilized by SmtB-SmtB bridge interactions. DNase I footprinting titrations also showed consecutive S1/S2 and S3/S4 SmtB occupancy. The footprints expand considerably in the presence of Zn2+. Hence, SmtB remains bound to the operator sites when Zn2+ ions are present. This result is further supported by gel retardation assay. Failure of the metal ions to dissociate SmtB from the DNA points to a hitherto unknown function of SmtB in the regulation of the smt locus.

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Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation , and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DELTADELTAG kcat/Km>2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 ANG) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.

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Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DELTAHD) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to DELTAHD, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DELTAHD value at 20degreeC and the heat capacity change (DELTACp) of dimer formation. In both NaCl and NaClO4, the obtained DELTACp value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DELTACp values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.

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The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

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Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of 15N- and 13C/15N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +- 0.11 and 1.02 +- 0.14 ANG, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by 1H and 13C chemical shifts and (15N)-1H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal

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Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>104-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >103-fold more active in the presence of 1.3 M Na2SO4. This activation is anion-dependent, following the Hofmeister series when normality is considered: SO42-apprxeqcitrate>Ac->Cl->Br->I-. The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na2SO4, and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P1 also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 mumol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.

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Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far- and near-UV circular dichroism spectroscopy, one-dimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

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Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg45 in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (KD=13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu367, Gly441, or Glu472 to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly441Lys showed markedly reduced binding to hEGF, implicating Gly441, in the L2 domain, as part of the binding site that recognizes Arg45 of hEGF.

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Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 ANG resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulf