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The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity. [ABSTRACT FROM AUTHOR]

Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by °35% were constructed. Only minor effects on kcat were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ≤30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-a,?-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The Ki for this adduct was °9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis. [ABSTRACT FROM AUTHOR]

The system specific chaperone DmsD interacts with the twin-arginine leader peptide of its substrate, DmsA, allowing for proper folding and assembly of the DmsA catalytic subunit of dimethyl sulfoxide reductase prior to translocation by the twin-arginine translocase. DmsD residues important for binding the complete 45-amino acid sequence of the DmsA leader (DmsAL) peptide were previously identified and found to cluster in a pocket of the DmsD structure. In this study, we have utilized isothermal titration calorimetry (ITC) to determine the dissociation constant and thermodynamic parameters of 15 single-substitution DmsD variant proteins and a synthetic DmsAL peptide consisting of 27 amino acids (DmsAL15-41). The stoichiometry values were determined via ITC, and the multimeric compositions of the DmsD variants in the absence and presence of peptide were characterized via size exclusion chromatography and native polyacrylamide gel electrophoresis. An up to 4-fold change in affinity was observed for DmsD variant proteins relative to that of wild-type DmsD, and variation of the entropic contribution to binding divided the binding site into two clusters: residues with either more or less favorable entropy. Substitution of hydrophobic residues along one helix face (helix 5) or prolines found on adjacent loops caused reduced binding affinity because of the increased entropic cost, which suggests that the twin-arginine motif of the DmsAL peptide binds to a preformed site on DmsD. Most DmsD variants were more than 90% monomeric in solution and bound a single peptide per protein molecule. The DmsD variant with the largest dimer population showed increased affinity and induced the formation of tetramers in the presence of peptide, suggesting that dimeric DmsD or an alternatively folded form of DmsD may play an as yet undefined role in binding. [ABSTRACT FROM AUTHOR]

β-2-Microglobulin (β2m) forms amyloid fibrils in the joints of patients undergoing hemodialysis treatment as a result of kidney failure. In the presence of stoichiometric amounts of Cu(II), β2m self-associates into discrete oligomeric species, including dimers, tetramers, and hexamers, before ultimately forming amyloid fibrils that contain no copper. To improve our understanding of whether Cu(II) is unique in its ability to induce film amyloid formation and to delineate the coordinative interactions that allow Cu(II) to exert its effect, we have examined the binding of Ni(II) and Zn(II) to film and the resulting influence that these metals have on β2m aggregation. We find that, in contrast to Cu(II), Ni(II) does not induce the oligomerization or aggregation of film, while Zn(II) promotes oligomerization but not amyloid fibril formation. Using X-ray absorption spectroscopy and new mass spectrometry-related techniques, we find that different binding modes are responsible for the different effects of Ni(II) and Zn(II). By comparing the binding modes of Cu(II) with Ni(II), we find that Cu(II) binding to Asp59 and the backbone amide between the first two residues of β2m are important for allowing the formation of amyloid-competent oligomers, as Ni(II) appears not to bind these sites on the protein. The oligomers formed in the presence of Zn(II) are permitted by this metal's ability to bridge two β2m units via His51. These oligomers, however, are not able to progress to form amyloid fibrils because Zn(II) does not induce the required structural changes near the N-terminus and His31. [ABSTRACT FROM AUTHOR]

The formation and stability of protein-protein interfaces are of obvious biological importance. While a large body of literature exists describing the effect of osmolytes on protein folding, very few studies address the effect of osmolytes on protein association and binding. The plant lectin concanavalin A (ConA), which undergoes a reversible tetramer-to-dimer equilibrium as a function of pH, was used as a model system to investigate the influence of nine osmolytes on protein self-association. The stabilizing or destabilizing impacts of the osmolytes were evaluated from pH titrations combined with circular dichroism spectroscopy. Relative to the dimer, trimethylamine N -oxide, betaine, proline, sarcosine, sorbitol, sucrose, and trehalose all stabilized the ConA tetramer to varying extents. Glycerol had a negligible effect, and urea destabilized the tetramer. From multiple titrations in different osmolyte concentrations, an m -value (a thermodynamic parameter describing the change in the association free energy per molar of osmolyte) was determined for each osmolyte. Experimental m -values were compared with those calculated using two theoretical models. The Tanford transfer model, with transfer free energies determined by Bolen and co-workers, failed to accurately predict the m -values in most cases. A model developed by Record and co-workers, currently applicable only to urea, betaine, and proline, more accurately predicted our experimental m -values, but significant discrepancies remained. Further theoretical work is needed to develop a thermodynamic model to predict the effect of osmolytes on protein-protein interfaces, and further experimental work is needed to determine if there is a general stabilization by osmolytes of such interfaces. [ABSTRACT FROM AUTHOR]

Biochemical and detailed structural information of human ether-ago- go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies. [ABSTRACT FROM AUTHOR]

α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule. [ABSTRACT FROM AUTHOR]

Glutaminase (GLS1/2) catalyzes the conversion of l-glutamine to l-glutamate and ammonia. The level of a splice variant of GLS1 (GAC) is elevated in certain cancers, and GAC is specifically inhibited by bis-2-(5-phenylacetimido-1,2,4,thiadiazol-2-yl)ethyl sulfide (BPTES). We report here the first full-length crystal structure of GAC in the presence and absence of BPTES molecules. Two BPTES molecules bind at an interface region of the GAC tetramer in a manner that appears to lock the GAC tetramer into a nonproductive conformation. The importance of these loops with regard to overall enzymatic activity of the tetramer was revealed by a series of GAC point mutants designed to create a BPTES resistant GAC. [ABSTRACT FROM AUTHOR]

Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of μOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity. [ABSTRACT FROM AUTHOR]

β-Amyloid (Aβ) is the main protein component of the amyloid plaques associated with Alzheimer's disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type (wt) TTR amyloid deposits are linked to senile systemic amyloidosis. a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from the toxic effects of Aβ deposition [Stein, T. D., and Johnson, J. A. (2002) J. Neurosci. 22, 7380-7388]. We examined the interaction of Aβ with wt TTR as well as two mutants: F87M/LI10M. engineered to be a stable monomer, and TI 19M. a naturally occurring mutant with a tetrameric stability higher than that of the wild type. On the basis of enzyme-linked immunoassays as well as cross-linking experiments, we conclude that Aβ monomers bind more to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aβ aggregates rather than Aβ monomers. Through tandem mass spectrometry analysis of cross-linked TTR-Aβ fragments, we identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation. TTR monomers decidedly arrest Aβ aggregate growth. These data provide important new insights into the nature of TTR- Aβ interactions. Such interactions may regulate TTR-mediated protection against Aβ toxicity. [ABSTRACT FROM AUTHOR]

A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with single-stranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coil and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where ∼70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB·ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA have no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to deterniine the dissociation constant for labeled SSB2·dT70 to be 1.1 μM at a high ionic strength (200mM NaCI). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced. [ABSTRACT FROM AUTHOR]

The M2 protein of influenza A viruses forms a tetrameric pH-activated proton-selective chennel that is targeted by the amantadine class of antiviral drugs. Its ion channel function has been extensively studied by electrophysiology and mutagenesis; however, the molecular mechanism of proton transport is still elusive, and the mechanism of inhibition by amantadine is controversial. We review the functional data on proton channel activity, molecular dynamics simulations of the proton conduction mechanism, and high-resolution structural and dynamical information of this membrane protein in lipid bilayers and lipid-mimetic detergents. These studies indicate that elucidation of the structural basis of M2 channel activity and inhibition requires thorough examination of the complex dynamics and conformational plasticity of the protein in different lipid bilayers and lipid-mimetic environments. [ABSTRACT FROM AUTHOR]

ABSTRACT: Hemoglobin (Hb) is an allosteric tetrameric protein made up of αβ heterodimers. The a and ,3 chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with [sup15]N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with ëhain selectivity by resonance Raman (RR) spectrcoscopy. For α but not for β chains, the frequency of the v[sub4] porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation ofproximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the a chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 ± 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the afi interface, evidence for which has emerged from UVRR studies of site mutants. [ABSTRACT FROM AUTHOR]

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of the KcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity of K+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by ∼103- or ∼104-fold for Ala and Cys substitution, respectively, consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature or Cu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations. [ABSTRACT FROM AUTHOR]

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS–PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 μmol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants. [ABSTRACT FROM AUTHOR]

Myc and Max belong to the b-HLH-LZ family of transcription factors. Heterodimerization between Myc and Max or homodimerization of Max allows these proteins to bind their cognate DNA sequence known as the E-box (CACGTG). Recent evidence has suggested that the c-Myc/Max heterodimeric b-HLH-LZ could interact to form a head-to-tail dimer of dimers and induce complex topologies such as loops in promoters containing more than one E-box sequence. In an attempt to shed light on this hypothesis, the interaction between the heterodimeric b-HLH-LZ of c-Myc/Max and a fragment of the hTERT promoter containing two E-box sequences was studied by atomic force microscopy. Specific binding events were observed at both E-box sites with equal probabilities. In accordance with previous results obtained by EMSA, we observed that the specific binding of the c-Myc/Max b-HLH-LZ bends the promoter. However no looping could be observed in a wide range of concentration encompassing the Ka (association constant) of the putative tetramer and the Ka1 for the specific binding of the heterodimer. In contrast, experiments performed with a mandatory c-Myc/Max b-HLH-LZ tetramer incubated with the hTERT promoter fragment allowed for the visualization of loops and cross-linked DNA strands originating from specific binding. Altogether, our results indicate that the c-Myc/Max b-HLH-LZ dimer binds specifically and equally to both E-box sites of the hTERT promoter and induces a significant bending of the promoter and that the suggested oligomerization of the c-Myc/Max heterodimeric b-HLH-LZ, if existing, is most likely too weak to induce the formation of a loop in a promoter. [ABSTRACT FROM AUTHOR]

Mdm2 negatively regulates p53 by inhibiting its transcriptional activity and promoting its degradation by functioning as an E3 ubiquitin ligase. The primary p53 binding site on mdm2 is located in its N-terminal domain. Through binding to p53 at its N-terminal transactivation domain, mdm2 directly blocks the transcriptional activation function of p53. We discovered that truncated mdm2 protein constructs without the N-terminal p53 binding domain are at least as active as full-length mdm2 in catalyzing p53 ubiquitination. Furthermore, the deletion of the central acidic domain significantly reduces the E3 ligase activity of mdm2 toward p53. We have also performed GST pull-down experiments to probe the direct binding of various mdm2 domain constructs toward full length p53 and found that mdm2 constructs without the N-terminal p53 binding domain retain the ability to bind to p53. Our kinetic and binding data localize the second p53 binding site between amino acids 211 and 361, including the acidic domain and the zinc finger region. Our work, consistent with other reports, suggests that the p53 tetramer interacts with at least two sites on mdm2. Although the interaction between the N-termini of mdm2 and p53 blocks the transactivation activity of p53, the interaction between the central domain of mdm2 and the core domain of p53 is critical for the ubiquitination and degradation of p53. This second mdm2–p53 interaction site represents an alternative target for small molecule modulators of the mdm2–p53 pathway. [ABSTRACT FROM AUTHOR]

We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (ΔHobs) and the negative heat capacity change (ΔCp,obs) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined ΔHobs over a much wider temperature range (5–60 °C) and as a function of monovalent salt concentration and type for SSB binding to (dT)70 under solution conditions that favor the fully wrapped (SSB)65 complex (monovalent salt concentration ≥0.20 M). Over this wider temperature range we observe a strongly temperature-dependent ΔCp,obs. The ΔHobs decreases as temperature increases from 5 to 35 °C (ΔCp,obs <0) but then increases at higher temperatures up to 60 °C (ΔCp,obs >0). Both salt concentration and anion type have large effects on ΔHobs and ΔCp,obs. These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 °C), the midpoint of which shifts to higher temperature (above 35 °C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br- > Cl- > F-, and these anions are then released upon binding ssDNA, affecting both ΔHobs and ΔCp,obs. We conclude that the experimentally measured values of ΔCp,obs for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (FGFs) play crucial roles in the regulation of key cellular processes such as angiogenesis, differentiation, and tumor growth. Suramin, a polysulfonated naphthylurea, is known to be a potent inhibitor of FGF-induced angiogenesis. Using isothermal titration calorimetry, we demonstrate that human acidic fibroblast growth factor (hFGF-1) binds to suramin with high affinity in the nanomolar range. The suramin:hFGF-1 binding stoichiometry is estimated to be 2:1. Size-exclusion chromatography data reveal that suramin oligomerizes hFGF-1 to form a stable tetramer. Thermal unfolding experiments monitored by steady state fluorescence, and limited trypsin digestion analysis data suggest that suramin- induced oligomerization of hFGF-1 occurs in two steps. The first step involves the binding of suramin at specific sites on the protein. Two molecules of suramin appear to bind simultaneously to one molecule of hFGF-1. Binding of suramin possibly involves formation of solvent-exposed nonpolar surfaces in hFGF-1. In the second step, FGF appears to oligomerize through coalescence of the solvent-accessible nonpolar surfaces. Results of the NMR experiments reveal that suramin binds to residues in the heparin binding pocket as well as to residues involved in FGF receptor binding. On the basis of the results of this study, we propose a model to explain the molecular mechanism(s) underlying the antimitogenic activity of suramin. To our knowledge, this is the first study in which suramin interaction sites on FGF have been characterized. [ABSTRACT FROM AUTHOR]

Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/ BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiq-uitination and E4 polyubiquitin ligases. [ABSTRACT FROM AUTHOR]

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)2 construct, affording (TTR-S-S-TTR)1 retaining a stable 16-stranded β-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)2 affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)2 construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)2 construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C2 axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof. [ABSTRACT FROM AUTHOR]

The X-ray crystal structures of Escherichia coli γ-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3221 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 Å resolution for the unliganded enzyme and 1.9 Å resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of γ-aminobutyrate aminotransferase to act on primary amine substrates (γ-aminobutyrate) in the first half-reaction and α-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the α-amino acid half-reaction, in which Arg398 binds the substrate α-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig γ-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed. [ABSTRACT FROM AUTHOR]

Previously, it was shown that the tetrameric potassium channel KcsA when present in a lipid bilayer can be dissociated by trifluoroethanol [van den Brink-van der Laan, E., et al. (2004) Biochemistry 43, 4240-4250]. Here, we demonstrate that this is a general property of small alcohols. We found that small alcohols dissociate the KcsA tetramer, at a concentration that depends on their membrane affinity. Importantly, the efficiency of the alcohol-induced tetramer dissociation was found to correlate with the efficiency of both alcohol-induced bilayer leakage and acyl chain disordering. Our data suggest that the ability of small alcohols to induce KcsA tetramer dissociation and to function as anesthetics depends on their effect on the membrane lateral pressure. [ABSTRACT FROM AUTHOR]

The potassium channel KcsA forms an extremely stable tetramer. Despite this high stability, it has been shown that the membrane-mimicking solvent 2,2,2-trifluoroethanol (TFE) can induce tetramer dissociation [Valiyaveetil, F. I., et al. (2002) Biochemistry 41, 10771-7, and Demmers, J. A. A., et al. (2003) FEBS Lett. 541, 69-77]. Here we have studied the effect of TFE on the structure and oligomeric state of the KcsA tetramer, reconstituted in different lipid systems. It was found that TFE changes the secondary and tertiary structure of KcsA and that it can dissociate the KcsA tetramer in all systems used. The tetramer is stabilized by a lipid bilayer as compared to detergent micelles. The extent of stabilization was found to depend on the nature of the lipids: a strong stabilizing effect of the nonbilayer lipid phosphatidylethaflolaltllne (PE) was observed, but no effect of the charged phoshosphatidylglycerol (PG) as compared to phosphatidylcholine (PC) was found. To understand how lipids stabilize KcsA against TFE-induced tetramer dissociation, we also studied the effect of TFE on the bilayer organization in the various lipid systems, using 31P and 2H NMR. The observed lipid dependency was similar as was found for tetramer stabilization: PE increased the bilayer stability as compared to PC, while PG behaved similar to PC. Furthermore, it was found that TFE has a large effect on the acyl chain ordering. The results indicate that TFE inserts primarily in the membrane interlace. We suggest that the lipid bilayer stabilizes the KcsA tetramer by the lateral pressure in the acyl chain region and that this stabilizing effect increases when a nonbilayer lipid like PE is present. [ABSTRACT FROM AUTHOR]

The stator of the bacterial flagellar motor is formed from the membrane proteins Moth and MotB, which associate in complexes with stoichiometry M0tA4MOtB2 (Kojima, S., and Blair, D. F., preceding paper in this issue). The MotAfMotB complexes conduct ions across the membrane, and couple ion flow to flagellar rotation by a mechanism that appears to involve conformational changes within the complex. Moth has four ernbrane-crossing segments, termed Al -A4, and MotB has one, termed B. We are studying the organization of the 18 membrane segments in the MotA4MotB2 complex by using targeted disulfide cross-linking. A previous cross-linking study showed that the two B segments in the complex (one from each MotB subunit) are arranged as a symmetrical dimer of ci-helices. Here, we extend the cross-linking study to segments A3 and A4. Single Cys residues were introduced by mutation in several consecutive positions in segments A3 and A4, and double mutants were made by pairwise combination of subsets of the Cys replacements in segments A3, A4, and B. Disulfide cross-linking of the single- and double-Cys proteins was studied in whole cells, in membranes, and in detergent solution. Several combinations of Cys residues in segments A3 and B gave a high yield of disulfide-linked MotA/ MotB heterodimer upon oxidation with iodine. Positions of efficient cross-linking identify a helix face on segment A3 that is in proximity to segment(s) B. Some combinations of Cys residues in segments A4 and B also gave a significant yield of disulfide-linked heterodimer, indicating that segment A4 is also near segment(s) B. Certain combinations of Cys residues in segments A3 and A4 cross-linked to form MotA tetramers in high yield upon oxidation. The high-yield positions identify faces on A3 and A4 that are at an interface between MotA subunits. [ABSTRACT FROM AUTHOR]

RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Nail. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase B acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase B forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase B is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D2 dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the Si -RNA binding domains of RNase B lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase EIRNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase B is the central component. [ABSTRACT FROM AUTHOR]

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and H, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structurefunction relationships. The binding modes of the substmte, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn[SUP2+] or Mg[SUP2+]) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme. [ABSTRACT FROM AUTHOR]

The core nucleosome, which comprises an H3-H4 tetramer and two H2A-H2B dimers, is not a static DNA packaging structure. The nucleosome is a dynamic protein-DNA complex, and the modulation of its structure is an important component of transcriptional regulation. To begin to understand the molecular details of nucleosome dynamics, we have investigated the stability of the isolated H2AH2B dimer. The urea-induced equilibrium responses of the heterodimer have been examined by far-UV circular dichroism and intrinsic tyrosine fluorescence. The two spectroscopic probes yielded coincident transitions, and global fitting of the reversible urea-induced unfolding further demonstrated that H2AH2B unfolds by a two-state equilibrium response. At physiological ionic strengths, the free energy of unfolding in the absence of urea of H2A-H2B is 11.8 ± 0.3 kcal mol[sup -1], moderate stability for a dimer of 26.4 kDa. The m value, or sensitivity of the unfolding to urea, is 2.9 ± 0.1 kcal mol[sup -1] M[sup -1]. This value is significantly larger than would be predicted for the unfolding of the dimerization motif alone (∼2 kcal mol[sup -1] M[sup -1]), suggesting that the N-terminal tails may adopt a collapsed, solvent-excluding structure that undergoes an unfolding transition. The efficacies of several potassium salts and three chloride salts to stabilize the H2A-H2B dimer were determined. The salt-dependent stabilization of the H2A-H2B dimer shows that the Hofmeister effect is the predominant mode of stabilization. However, studies employing multiple salts suggest that there is a component of stabilization that must arise from screening of electrostatic repulsion in the highly basic heterodimer. The most highly charged regions of the dimer are the N-terminal tails, sites of posttranslational modifications such as acetylation and phosphorylation. These modifications, which alter the charge density of the tails, are involved in regulation of nucleosome dynamics. [ABSTRACT FROM AUTHOR]

Examines transposing sequences between fetal and adult hemoglobins. Recombinant analysis of the adult hemoglobin/fetal hemoglobin systems; Relationship between allosteric, sliding interface and effects of allosteric regulator in promoting oxygen release; Differences in tetramer-dimer dissociation constants of adult and fetal tetramers.

Discusses the effects of mutations in apyrase conserved region determined by whether they occur in intact tetrameres or in disrupted tetrameres or monomers. Result of substitution in tetrameres lacking either transmembrane domain and in monomers lacking transmembrane domains; Varied adenosine triphosphatase and adenosine diphosphatase mechanisms used by CD39.

Examines the pathogenicity of L55P transthyretin (TTR) under acidic condition and photofilament under physiological condition. Difference of L55P-TTR from other TTR variants; Mechanisms of L55P-TTR tetramer dissociation; Activation barrier for L55P-TTR dissociation; Sensitivity of L55P-TTR variant tetramer to acidification and protein concentration.

Studies the thermal stability and mode of oligomerization of the homotetrameric legume lectin, peanut agglutinin. Use of differential scanning calorimetry; Reversibility of unfolding process;Effect of presence of saturating amounts of carbohydrate ligands; Folded monomer as the intermediate phase in the unfolding of the agglutinin.

Presents a study which examined the implications of tetramer formation by retinoid X receptor (RXR), a nuclear receptor which is activated by 9-cis-retinoic acid (9cRA), for the ability of the receptor to heterodimerize with receptors RAR and VDR. Effects of cognate ligands on heterodimerization of RXR with receptors; Examination of the oligomerization behavior and transcriptional activities of two site specific mutants of mRXR...

Provides information on a study investigating the structures of the UCCG and UGCG tetraloops formed in octamer ribonucleotide hairpin sequences. Methodology and materials used to conduct the study; Information on vibrational conformational markers; Phosphate backbone markers; Findings of the study.

p53 is a tetrameric multidomain protein that triggers the anticancer cellular response to stress. We have calculated a three-dimensional reconstruction of full-length human p53 in the absence of DNA using single-particle electron microscopy. The reconstruction of DNA-free full-length p53 shows a square-shaped structure with four distinct domains and a hollow center. In comparison with the known compacted DNA-bound full-length p53 structures, the DNA-free p53 tetramer adopts a relaxed conformation with separated monomers and oligomerization interfaces different from those of the DNA-bound conformation. [ABSTRACT FROM AUTHOR]