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Dimer Interface (dimer + interface)

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Chemistry100%

Selected Abstracts

Functional role of fumarate site Glu59 involved in allosteric regulation and subunit,subunit interaction of human mitochondrial NAD(P)+ -dependent malic enzyme

FEBS JOURNAL, Issue 4 2009
Ju-Yi Hsieh
Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+ -dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and ,KA,Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The KA,Fum and ,KA,Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer,tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer,tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP+ -dependent malic enzyme and cytosolic NADP+ -dependent malic enzyme in fumarate activation and malate cooperativity. [source]

Crystal structure of a staphylokinase variant

FEBS JOURNAL, Issue 2 2002
A model for reduced antigenicity
Staphylokinase (SAK) is a 15.5-kDa protein from Staphylococcus aureus that activates plasminogen by forming a 1 : 1 complex with plasmin. Recombinant SAK has been shown in clinical trials to induce fibrin-specific clot lysis in patients with acute myocardial infarction. However, SAK elicits high titers of neutralizing antibodies. Biochemical and protein engineering studies have demonstrated the feasibility of generating SAK variants with reduced antigenicity yet intact thrombolytic potency. Here, we present X-ray crystallographic evidence that the SAK(S41G) mutant may assume a dimeric structure. This dimer model, at 2.3-Å resolution, could explain a major antigenic epitope (residues A72,F76 and residues K135-K136) located in the vicinity of the dimer interface as identified by phage-display. These results suggest that SAK antigenicity may be reduced by eliminating dimer formation. We propose several potential mutation sites at the dimer interface that may further reduce the antigenicity of SAK. [source]

The oligomerization of CynR in Escherichia coli

PROTEIN SCIENCE, Issue 11 2009
Gwendowlyn S. Knapp
Abstract Deletion analysis and alanine-scanning based on a homology-based interaction model were used to identify determinants of oligomerization in the transcriptional regulator CynR, a member of the LysR-type transcriptional regulator (LTTR) family. Deletion analysis confirmed that the putative regulatory domain of CynR was essential for driving the oligomerization of , repressor-CynR fusion proteins. The interaction surface of a different LTTR and OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface, which were targeted for alanine-scanning mutagenesis. Oligomerization was assayed by the ability of mutant , repressor-CynR fusions to assemble in E. coli revealing interesting similarities and differences between OxyR and CynR. [source]

L2, loop is critical for caspase-7 active site formation

PROTEIN SCIENCE, Issue 7 2009
Witold A. Witkowski
Abstract The active sites of caspases are composed of four mobile loops. A loop (L2) from one half of the dimer interacts with a loop (L2,) from the other half of the dimer to bind substrate. In an inactive form, the two L2, loops form a cross-dimer hydrogen-bond network over the dimer interface. Although the L2, loop has been implicated as playing a central role in the formation of the active-site loop bundle, its precise role in catalysis has not been shown. A detailed understanding of the active and inactive conformations is essential to control the caspase function. We have interrogated the contributions of the residues in the L2, loop to catalytic function and enzyme stability. In wild-type and all mutants, active-site binding results in substantial stabilization of the complex. One mutation, P214A, is significantly destabilized in the ligand-free conformation, but is as stable as wild type when bound to substrate, indicating that caspase-7 rests in different conformations in the absence and presence of substrate. Residues K212 and I213 in the L2, loop are shown to be essential for substrate-binding and thus proper catalytic function of the caspase. In the crystal structure of I213A, the void created by side-chain deletion is compensated for by rearrangement of tyrosine 211 to fill the void, suggesting that the requirements of substrate-binding are sufficiently strong to induce the active conformation. Thus, although the L2, loop makes no direct contacts with substrate, it is essential for buttressing the substrate-binding groove and is central to native catalytic efficiency. [source]

Structure and heme binding properties of Escherichia coli O157:H7 ChuX

PROTEIN SCIENCE, Issue 4 2009
Michael D. L. Suits
Abstract For many pathogenic microorganisms, iron acquisition from host heme sources stimulates growth, multiplication, ultimately enabling successful survival and colonization. In gram-negative Escherichia coli O157:H7, Shigella dysenteriae and Yersinia enterocolitica the genes encoded within the heme utilization operon enable the effective uptake and utilization of heme as an iron source. While the complement of proteins responsible for heme internalization has been determined in these organisms, the fate of heme once it has reached the cytoplasm has only recently begun to be resolved. Here we report the first crystal structure of ChuX, a member of the conserved heme utilization operon from pathogenic E. coli O157:H7 determined at 2.05 Å resolution. ChuX forms a dimer which remarkably given low sequence homology, displays a very similar fold to the monomer structure of ChuS and HemS, two other heme utilization proteins. Absorption spectral analysis of heme reconstituted ChuX demonstrates that ChuX binds heme in a 1:1 manner implying that each ChuX homodimer has the potential to coordinate two heme molecules in contrast to ChuS and HemS where only one heme molecule is bound. Resonance Raman spectroscopy indicates that the heme of ferric ChuX is composed of a mixture of coordination states: 5-coordinate and high-spin, 6-coordinate and low-spin, and 6-coordinate and high-spin. In contrast, the reduced ferrous form displays mainly a 5-coordinate and high-spin state with a minor contribution from a 6-coordinate and low-spin state. The ,Fe-CO and ,C-O frequencies of ChuX-bound CO fall on the correlation line expected for histidine-coordinated hemoproteins indicating that the fifth axial ligand of the ferrous heme is the imidazole ring of a histidine residue. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuX to probe the heme binding sites and dimer interface. Spectral analysis of ChuX and mutants suggests involvement of H65 and H98 in heme coordination as mutations of both residues were required to abolish the formation of the hexacoordination state of heme-bound ChuX. [source]

Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function

PROTEIN SCIENCE, Issue 5 2008
Shira Albeck
Abstract Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 Å resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 5,-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az. [source]

N15 Cro and , Cro: Orthologous DNA-binding domains with completely different but equally effective homodimer interfaces

PROTEIN SCIENCE, Issue 5 2008
Matthew S. Dubrava
Abstract Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 Å resolution. The subunit fold contains five ,-helices and is closely similar to the structure of P22 Cro (1.3 Å backbone room mean square difference over 52 residues), but quite different from that of , Cro, a structurally diverged member of this family with a mixed ,-helix/,-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 Å2 change in accessible surface area per dimer) and also dimerizes in solution with a Kd of 5.1 ± 1.5 ,M. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of , Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms. [source]

Crystal structure of an enhancer of rudimentary homolog (ERH) at 2.1 Å resolution

PROTEIN SCIENCE, Issue 7 2005
Ryoichi Arai
Abstract The enhancer of rudimentary gene, e(r), of Drosophila melanogaster encodes an enhancer of rudimentary (ER) protein with functions implicated in pyrimidine biosynthesis and the cell cycle. The ER homolog (ERH) is highly conserved among vertebrates, invertebrates, and plants. Xenopus laevis ERH was reported to be a transcriptional repressor. Here we report the 2.1 Å crystal structure of murine ERH (Protein Data Bank ID 1WZ7), determined by the multiwavelength anomalous dispersion (MAD) method. The monomeric structure of ERH comprises a single domain consisting of three ,-helices and four ,-strands, which is a novel fold. In the crystal structure, ERH assumes a dimeric structure, through interactions between the ,-sheet regions. The formation of an ERH dimer is consistent with the results of analytical ultracentrifugation. The residues at the core region and at the dimer interface are highly conserved, suggesting the conservation of the dimer formation as well as the monomer fold. The long flexible loop (44,53) is also significantly conserved, suggesting that this loop region may be important for the functions of ERH. In addition, the putative phosphorylation sites are located at the start of the ,2-strand (Thr18) and at the start of the ,1-helix (Ser24), implying that the phosphorylation might cause some structural changes. [source]

Expansion of the aspartate ,-semialdehyde dehydrogenase family: the first structure of a fungal ortholog

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010
Buenafe T. Arachea
The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development. [source]

Structures of the apo and holo forms of formate dehydrogenase from the bacterium Moraxella sp.

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009
C-1: towards understanding the mechanism of the closure of the interdomain cleft
NAD+ -dependent formate dehydrogenase (FDH) catalyzes the oxidation of formate ion to carbon dioxide coupled with the reduction of NAD+ to NADH. The crystal structures of the apo and holo forms of FDH from the methylotrophic bacterium Moraxella sp. C-1 (MorFDH) are reported at 1.96 and 1.95,Å resolution, respectively. MorFDH is similar to the previously studied FDH from the bacterium Pseudomonas sp. 101 in overall structure, cofactor-binding mode and active-site architecture, but differs in that the eight-residue-longer C-terminal fragment is visible in the electron-density maps of MorFDH. MorFDH also differs in the organization of the dimer interface. The holo MorFDH structure supports the earlier hypothesis that the catalytic residue His332 can form a hydrogen bond to both the substrate and the transition state. Apo MorFDH has a closed conformation of the interdomain cleft, which is unique for an apo form of an NAD+ -dependent dehydrogenase. A comparison of the structures of bacterial FDH in open and closed conformations allows the differentiation of the conformational changes associated with cofactor binding and domain motion and provides insights into the mechanism of the closure of the interdomain cleft in FDH. The C-terminal residues 374,399 and the substrate (formate ion) or inhibitor (azide ion) binding are shown to play an essential role in the transition from the open to the closed conformation. [source]

Structure of NS1A effector domain from the influenza A/Udorn/72 virus

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009
Shuangluo Xia
The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains: an N-terminal 73-amino-acid RNA-binding domain and a C-terminal effector domain. Here, the crystallographic structure of the NS1A effector domain of influenza A/Udorn/72 virus is presented. Structure comparison with the NS1 effector domain from mouse-adapted influenza A/Puerto Rico/8/34 (PR8) virus strain reveals a similar monomer conformation but a different dimer interface. Further analysis and evaluation shows that the dimer interface observed in the structure of the PR8 NS1 effector domain is likely to be a crystallographic packing effect. A hypothetical model of the intact NS1 dimer is presented. [source]

Structural asymmetry and intersubunit communication in muscle creatine kinase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2007
Jeffrey F. Ohren
The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65,Å resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation. [source]

Ligand binding at the transthyretin dimer,dimer interface: structure of the transthyretin,T4Ac complex at 2.2,Å resolution

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2005
Vivian Cody
The crystal structure of the complex of human transthyretin (hTTR) with 3,3,,5,5,-tetraiodothyroacetic acid (T4Ac) has been determined to 2.2,Å resolution. The complex crystallizes in the orthorhombic space group P21212, with unit-cell parameters a = 43.46, b = 85.85, c = 65.44,Å. The structure was refined to R = 17.3% and Rfree = 21.9% for reflections without any ,-cutoff. T4Ac is bound in both the forward and the reverse mode in the two binding sites of hTTR. In the forward orientation, T4Ac binds in a position similar to that described for thyroxine (T4) in the orthorhombic hTTR,T4 complex. In this orientation, the iodine substituents of the phenolic ring are bound in the P3,/P2 halogen pockets. In the reverse orientation, which is the major binding mode of T4Ac, the ligand is bound deep in the TTR channel, with the carboxylic group bound in the P3, pocket and forming simultaneous polar interactions with the residues constituting the two hormone-binding sites. Such interactions of a thyroxine-analogue ligand bound in the reverse mode have never been observed in TTR complexes previously. [source]

Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2005
Pascal Retailleau
Urate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135,kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-azaxanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3121, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P21 with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P21212 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (,1.6,Å resolution compared with 2.3,3.2,Å for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors. [source]

Structure of cyclized green fluorescent protein

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2002
Andreas Hofmann
Crystals of cyclic green fluorescent protein (cGFP) engineered by the previously reported split intein technology [Iwai et al. (2001), J. Biol. Chem.276, 16548,16554] were obtained and the structure was solved using molecular replacement. Although the core of the protein can unambiguously be fitted from the first to the last residue of the genuine sequence, the electron density in the region of the linker peptide is rather poor owing to the high water content of the crystals. Therefore, it is concluded that this part of the protein is highly disordered in the present structure and is very flexible. This is supported by the absence of crystal contacts in the linker-peptide region and the fact that the core of the protein exhibits a very similar conformation to that known from other GFP structures, thereby not implicating any constraints arising from the presence of the artificial linker. Nevertheless, the density is consistent with the loop being intact, as confirmed by mass spectroscopy of dissolved crystals. The present structure contains an antiparallel cGFP dimer where the dimer interface is clearly different from other crystal structures featuring two GFP molecules. This adds further support to the fact that the cylinder surface of GFP is rather versatile and can employ various polar and non-polar patches in protein,protein interactions. [source]

Metal-free MIRAS phasing: structure of apo-S100A3

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002
Peer R. E. Mittl
S100 proteins are involved in metal-dependent intracellular signalling. Metal-free S100A3, a cysteine-rich Ca2+ - and Zn2+ -­binding protein, has been crystallized by vapour diffusion under the strict exclusion of oxygen and in the absence of divalent metal ions. Metal binding induces large conformational changes, rendering the apo-S100A3 crystals very sensitive to various metal compounds. Therefore, the structure was solved by MIRAS phasing using potassium iodide and xenon derivatives. Iodide replaces a water molecule at the surface of the S100A3 protein, whereas xenon binds in a hydrophobic cavity at the dimer interface. Despite significant non-isomorphism, the combination of both derivatives was sufficient for structure determination. The overall apo-S100A3 structure resembles the structures of metal-free S100B and S100A6 solution structures. In contrast to the NMR structures, the EF-hand loops are well ordered in the apo-S100A3 crystal structure. In the N-terminal pseudo-EF-­hand loop a water molecule occupies the position of the Ca2+ ion. The C-terminal canonical EF-hand loop shows an extended conformation and a different helix arrangement to other S100/metal complex crystal structures. [source]

Structure of a Nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit ,-­sheets

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2002
Shuishu Wang
Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the ,-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved ,-sheet of 11 ,-strands and two ,-helices, forming an ,,,,, sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first ,-helix and the ,-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein. [source]

The three-dimensional structure of cytosolic bovine retinal creatine kinase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2001
Dominic Tisi
Creatine kinase (CK) catalyses the reversible transfer of the phosphate moiety from phosphocreatine (PCr) to ADP, generating creatine and ATP. The crystal structure of a cytosolic brain-type creatine kinase is reported at 2.3,Å. The biological dimer sits on a crystallographic twofold axis. The N-­terminal residues of both subunits come very close to the crystallographic twofold at the dimer interface. The electron density observed is consistent with two alternative conformations for the N-termini, as previously found for chicken brain-type creatine kinase. [source]

The 1.9,Å structure of the branched-chain amino-acid transaminase (IlvE) from Mycobacterium tuberculosis

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 11 2009
L. W. Tremblay
Unlike mammals, bacteria encode enzymes that synthesize branched-chain amino acids. The pyridoxal 5,-phosphate-dependent transaminase performs the final biosynthetic step in these pathways, converting keto acid precursors into ,-amino acids. The branched-chain amino-acid transaminase from Mycobacterium tuberculosis (MtIlvE) has been crystallized and its structure has been solved at 1.9,Å resolution. The MtIlvE monomer is composed of two domains that interact to form the active site. The biologically active form of IlvE is a homodimer in which each monomer contributes a substrate-specificity loop to the partner molecule. Additional substrate selectivity may be imparted by a conserved N-terminal Phe30 residue, which has previously been observed to shield the active site in the type IV fold homodimer. The active site of MtIlvE contains density corresponding to bound PMP, which is likely to be a consequence of the presence of tryptone in the crystallization medium. Additionally, two cysteine residues are positioned at the dimer interface for disulfide-bond formation under oxidative conditions. It is unknown whether they are involved in any regulatory activities analogous to those of the human mitochondrial branched-chain amino-acid transaminase. [source]

Monellin (MNEI) at 1.15,Å resolution

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2007
J. R. Hobbs
The X-ray crystal structure of a single-chain monellin protein (MNEI) has been determined at 1.15,Å resolution. The model was refined to convergence employing anisotropic displacement parameters and riding H atoms to produce a final model with Rwork and Rfree values of 0.132 and 0.162, respectively. The crystal contains a single MNEI protein in the asymmetric unit and unusually lacks the dimer interface observed in all previous crystal structures of monellin and its single-chain derivatives. The high resolution allowed a more detailed view of MNEI than previously possible, with 38 of the 96 residues modelled with alternative side-chain conformations, including four core residues Thr12, Cys41, Leu62 and Ile75. Four stably bound negative ions were also located, providing new insight into potential electrostatic interactions of MNEI with the largely negatively charged surface of the sweet taste receptor T1R2,T1R3. [source]

The quaternary structure of the amidase from Geobacillus pallidus RAPc8 is revealed by its crystal packing

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2006
Vinod B. Agarkar
The amidase from Geobacillus pallidus RAPc8, a moderate thermophile, is a member of the nitrilase enzyme superfamily. It converts amides to the corresponding acids and ammonia and has application as an industrial catalyst. RAPc8 amidase has been cloned and functionally expressed in Escherichia coli and has been purified by heat treatment and a number of chromatographic steps. The enzyme was crystallized using the hanging-drop vapour-diffusion method. Crystals produced in the presence of 1.2,M sodium citrate, 400,mM NaCl, 100,mM sodium acetate pH 5.6 were selected for X-ray diffraction studies. A data set having acceptable statistics to 1.96,Å resolution was collected under cryoconditions using an in-house X-ray source. The space group was determined to be primitive cubic P4232, with unit-cell parameter a = 130.49 (±0.05) Å. The structure was solved by molecular replacement using the backbone of the hypothetical protein PH0642 from Pyrococcus horikoshii (PDB code 1j31) with all non-identical side chains substituted with alanine as a probe. There is one subunit per asymmetric unit. The subunits are packed as trimers of dimers with D3 point-group symmetry around the threefold axis in such a way that the dimer interface seen in the homologues is preserved. [source]

Structure of human salivary ,-amylase crystallized in a C -centered monoclinic space group

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2006
S. Zoë Fisher
Human salivary ,-amylase (HSA) is a major secretory protein component of saliva and has important biological functions, including the initial digestion of starch. HSA acts as a monomer and mediates the hydrolysis of ,-1,4-glucosidic linkages in oligosaccharides. To date, all published crystal structures of HSA have been crystallized as monomers in space group P212121. Here, the serendipitous purification, crystallization and ultimate structure determination of a HSA non-crystallographic symmetry (NCS) dimer, while attempting to purify human carbonic anhydrase VI (HCA VI) from saliva using an affinity resin for ,-class carbonic anhydrases, is presented. On further investigation, it was shown that HSA could only be copurified using the affinity resin in the presence of HCA VI which is glycosylated and not the non-glycosylated HCA II. The identification of the HSA crystals was carried out by peptide mapping and mass spectrometry. HSA was shown to have crystallized as an NCS dimer in space group C2, with unit-cell parameters a = 150.9, b = 72.3, c = 91.3,Å, , = 102.8°. The NCS dimer crystal structure is reported to 3.0,Å resolution, with a refined Rcryst of 0.228. The structure is compared with the previously reported P212121 monomer structures and the crystal packing and dimer interface are discussed. [source]

X-ray structure of glutathione S -transferase from Schistosoma japonicum in a new crystal form reveals flexibility of the substrate-binding site

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2005
Arne Christian Rufer
The crystal structure of the 26,kDa glutathione S -transferase from Schistosoma japonicum (SjGST) was determined at 3,Å resolution in the new space group P212121. The structure of orthorhombic SjGST reveals unique features of the ligand-binding site and dimer interface when compared with previously reported structures. SjGST is recognized as the major detoxification enzyme of S. japonicum, a pathogenic helminth causing schistosomiasis. As resistance against the established inhibitor of SjGST, praziquantel, has been reported these results might prove to be valuable for the development of novel drugs. [source]

N15 Cro and , Cro: Orthologous DNA-binding domains with completely different but equally effective homodimer interfaces

PROTEIN SCIENCE, Issue 5 2008
Matthew S. Dubrava
Abstract Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 Å resolution. The subunit fold contains five ,-helices and is closely similar to the structure of P22 Cro (1.3 Å backbone room mean square difference over 52 residues), but quite different from that of , Cro, a structurally diverged member of this family with a mixed ,-helix/,-sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 Å2 change in accessible surface area per dimer) and also dimerizes in solution with a Kd of 5.1 ± 1.5 ,M. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self-associate detectably in solution. Instead, the level of self-association and interfacial area for N15 Cro is similar to that of , Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all-helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms. [source]

Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis

PROTEIN SCIENCE, Issue 8 2007
Nicole A. Kruh
Abstract The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis (FAS-II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH-NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH-NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS-II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein,protein interactions involving InhA and how these ligand-induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild-type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross-linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross-linking across one of the dimer,dimer interfaces in the InhA tetramer. Interestingly, a similar ligand-induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present. [source]