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Subunit Interface (subunit + interface)
Selected AbstractsArginine-induced conformational change in the c -ring/a -subunit interface of ATP synthaseFEBS JOURNAL, Issue 9 2008Thomas Vorburger The rotational mechanism of ATP synthases requires a unique interface between the stator a subunit and the rotating c -ring to accommodate stability and smooth rotation simultaneously. The recently published c -ring crystal structure of the ATP synthase of Ilyobacter tartaricus represents the conformation in the absence of subunit a. However, in order to understand the dynamic structural processes during ion translocation, studies in the presence of subunit a are required. Here, by intersubunit Cys,Cys cross-linking, the relative topography of the interacting helical faces of subunits a and c from the I. tartaricus ATP synthase has been mapped. According to these data, the essential stator arginine (aR226) is located between the c -ring binding pocket and the cytoplasm. Furthermore, the spatially vicinal residues cT67C and cG68C in the isolated c -ring structure yielded largely asymmetric cross-linking products with aN230C of subunit a, suggesting a small, but significant conformational change of binding-site residues upon contact with subunit a. The conformational change was dependent on the positive charge of the stator arginine or the aR226H substitution. Energy-minimization calculations revealed possible modes for the interaction between the stator arginine and the c -ring. These biochemical results and structural restraints support a model in which the stator arginine operates as a pendulum, moving in and out of the binding pocket as the c -ring rotates along the interface with subunit a. This mechanism allows efficient interaction between subunit a and the c- ring and simultaneously allows almost frictionless movement against each other. [source] Physico-chemical properties of molten dimer ascorbate oxidaseFEBS JOURNAL, Issue 22 2006Eleonora Nicolai The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri-nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH-dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra-subunit electrostatic interactions. The dimeric species is present even at 3 × 108 Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (,V , ,200 mL·mol,1) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (,G , 2 kcal·mol,1) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes. [source] Integrin ,IIb,3:ligand interactions are linked to binding-site remodelingPROTEIN SCIENCE, Issue 8 2006Roy R. Hantgan Abstract This study tested the hypothesis that high-affinity binding of macromolecular ligands to the ,IIb,3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between ,IIb,3 and two model ligands,echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's ,-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site,were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the ,IIb/,3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming,i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand-mimetic peptide cHarGD,promotes complex formation with fibrinogen's ,-module. We also observed that the ,-module's flexible carboxy terminus was not required for ,IIb,3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers. [source] Allosteric transition pathways in the lactose repressor protein core domains: Asymmetric motions in a homodimerPROTEIN SCIENCE, Issue 11 2003Terence C. Flynn Abstract The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain ,-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer,monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; ,-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer,monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data. [source] A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactionsACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Bret D. Freudenthal Eukaryotic proliferating cell nuclear antigen (PCNA) is an essential replication accessory factor that interacts with a variety of proteins involved in DNA replication and repair. Each monomer of PCNA has an N-terminal domain A and a C-terminal domain B. In the structure of the wild-type PCNA protein, domain A of one monomer interacts with domain B of a neighboring monomer to form a ring-shaped trimer. Glu113 is a conserved residue at the subunit interface in domain A. Two distinct X-ray crystal structures have been determined of a mutant form of PCNA with a substitution at this position (E113G) that has previously been studied because of its effect on translesion synthesis. The first structure was the expected ring-shaped trimer. The second structure was an unanticipated nontrimeric form of the protein. In this nontrimeric form, domain A of one PCNA monomer interacts with domain A of a neighboring monomer, while domain B of this monomer interacts with domain B of a different neighboring monomer. The B,B interface is stabilized by an antiparallel ,-sheet and appears to be structurally similar to the A,B interface observed in the trimeric form of PCNA. The A,A interface, in contrast, is primarily stabilized by hydrophobic interactions. Because the E113G substitution is located on this hydrophobic surface, the A,A interface should be less favorable in the case of the wild-type protein. This suggests that the side chain of Glu113 promotes trimer formation by destabilizing these possible alternate subunit interactions. [source] The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2007Ella Czarina Magat Juan The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9,Å resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit,subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products. [source] Structure of Staphylococcus aureus adenylosuccinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discoveryACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2010Paul K. Fyfe The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels. As such, the enzyme is predicted to be essential for the survival of S. aureus and to be a potential therapeutic target. Comparisons of this pathogen PurB with the enzyme from Escherichia coli are presented to allow discussion concerning the enzyme mechanism. Comparisons with human PurB suggest that the close similarity of the active sites would make it difficult to identify species-specific inhibitors for this enyme. However, there are differences in the way that the subunits are assembled into dimers. The distinct subunit,subunit interfaces may provide a potential area to target by exploiting the observation that creation of the enzyme active site is dependent on oligomerization. [source] An orthorhombic form of Escherichia coli aminopeptidase P at 2.4,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2003Stephen C. Graham Aminopeptidase P (AMPP) from Escherichia coli cleaves the N-terminal residue from an oligopeptide if the second residue is proline. The active site contains a dinuclear metal centre. Following earlier structural analyses of crystals in space groups P6422 and I4122, the structure of AMPP has been solved and refined in the orthorhombic space group C2221 at 2.4,Å resolution. There are six subunits in the asymmetric unit. These are arranged in two types of tetramer. One tetramer comprises four crystallographically independent subunits, while the other comprises two pairs of subunits related by a crystallographic twofold axis. The final model of 20,994 protein atoms, 1618 water molecules and 12 metal atoms refined to residuals R = 0.195 and Rfree = 0.215. The molecular structure confirms most of the previously reported features, including the subunit,subunit interfaces in the tetramer and persistent disorder at some residues. The metal,ligand bond lengths at the active site suggest that one of the two Mn atoms is five-coordinate rather than six-coordinate. [source] The 1.30,Å resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimerACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000Jane E. Ladner The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30,Å resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P212121 with unit-cell parameters a = 52.2, b = 83.8, c = 86.0,Å. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol.240, 476,500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an Rfree of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice. [source] | ||||||||||