Resolution Structure (resolution + structure)

Distribution by Scientific Domains
Distribution within Chemistry

Kinds of Resolution Structure

  • atomic resolution structure


  • Selected Abstracts


    High resolution structure and catalysis of O -acetylserine sulfhydrylase isozyme B from Escherichia coli

    FEBS JOURNAL, Issue 20 2007
    Georg Zocher
    The crystal structure of the dimeric O -acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O -acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O -acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction. [source]


    High resolution structure of the HDGF PWWP domain: A potential DNA binding domain

    PROTEIN SCIENCE, Issue 2 2006
    Stephen M. Lukasik
    Abstract Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. This 70,amino acid motif, originally identified from the WHSC1 gene, has been found in more than 60 eukaryotic proteins. In addition to the PWWP domain, many proteins in this class contain known chromatin remodeling domains, suggesting a role for HDGF in chromatin remodeling. We have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a nonspecific DNA-binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA. [source]


    Topography of a 2.0 Å structure of ,1 -antitrypsin reveals targets for rational drug design to prevent conformational disease

    PROTEIN SCIENCE, Issue 7 2000
    Peter R. Elliott
    Abstract Members of the serpin family of serine proteinase inhibitors play important roles in the inflammatory, coagulation, fibrinolytic, and complement cascades. An inherent part of their function is the ability to undergo a structural rearrangement, the stressed (S) to relaxed (R) transition, in which an extra strand is inserted into the central A ,-sheet. In order for this transition to take place, the A sheet has to be unusually flexible. Malfunctions in this flexibility can lead to aberrant protein linkage, serpin inactivation, and diseases as diverse as cirrhosis, thrombosis, angioedema, emphysema, and dementia. The development of agents that control this conformational rearrangement requires a high resolution structure of an active serpin. We present here the topology of the archetypal serpin ,1 -antitrypsin to 2 Å resolution. This structure allows us to define five cavities that are potential targets for rational drug design to develop agents that will prevent conformational transitions and ameliorate the associated disease. [source]


    Atomic resolution structure of pseudoazurin from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans: structural insights into its spectroscopic properties

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009
    Daisuke Hira
    The crystal structure of native pseudoazurin (HdPAz) from the methylotrophic denitrifying bacterium Hyphomicrobium denitrificans has been determined at a resolution of 1.18,Å. After refinement with SHELX employing anisotropic displacement parameters and riding H atoms, Rwork and Rfree were 0.135 and 0.169, respectively. Visualization of the anisotropic displacement parameters as thermal ellipsoids provided insight into the atomic motion within the perturbed type 1 Cu site. The asymmetric unit includes three HdPAz molecules which are tightly packed by head-to-head cupredoxin dimer formation. The shape of the Cu-atom ellipsoid implies significant vibrational motion diagonal to the equatorial xy plane defined by the three ligands (two His and one Cys). The geometric parameters of the type 1 Cu site in the HdPAz structure differ unambiguously from those of other pseudoazurins. It is demonstrated that their structural aspects are consistent with the unique visible absorption spectrum. [source]


    The 1.25,Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2008
    Farah Javid-Majd
    Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79,Å. The structure of the apoenzyme reveals that the protein is composed of five ,-helices with connecting loops and is a member of the ,-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between ,-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily. [source]


    Modulation of activity by Arg407: structure of a fungal ,-1,2-mannosidase in complex with a substrate analogue

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2008
    Yuri D. Lobsanov
    Class I ,-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95,Å resolution structure of a fungal ,-1,2-mannosidase in complex with the substrate analogue methyl-,- d -lyxopyranosyl-(1,,2)-,- d -mannopyranoside (LM) shows the intact disaccharide spanning the ,1/+1 subsites, with the d -lyxoside ring in the ,1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5, hydroxymethyl group on the d -lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the d -lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily. [source]


    The 1.9,Å resolution structure of Mycobacterium tuberculosis 1-deoxy- d -xylulose 5-­phosphate reductoisomerase, a potential drug target

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2006
    Lena M. Henriksson
    1-Deoxy- d -xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-­deoxy- d -xylulose 5-phosphate to form 2- C -methyl- d -­erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms. The pathway is not found in humans; the mevalonate pathway is instead used for the formation of isopentenyl diphosphate. This difference, combined with its essentiality, makes the reductoisomerase an excellent drug target in a number of pathogenic organisms. The structure of 1-deoxy- d -xylulose 5-phosphate reductoisomerase from Mycobacterium tuberculosis (Rv2870c) was solved by molecular replacement and refined to a resolution of 1.9,Å. The enzyme exhibited an estimated kcat of 5.3,s,1 and Km and kcat/Km values of 7.2,µM and 7.4 × 105,M,1,s,1 for NADPH and 340,µM and 1.6 × 104,M,1,s,1 for 1-deoxy- d -­xylulose 5-phosphate. In the structure, a sulfate is bound at the expected site of the phosphate moiety of the sugar substrate. The M. tuberculosis enzyme displays a similar fold to the previously published structures from Escherichia coli and Zymomonas mobilis. Comparisons offer suggestions for the design of specific drugs. Furthermore, the new structure represents an intermediate conformation between the open apo form and the closed holo form observed previously, giving insights into the conformational changes associated with catalysis. [source]


    Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75,Å

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2006
    Markus Linder
    Hydrophobins are small proteins secreted by filamentous fungi that have a unique ability to spontaneously form amphiphilic layers. Hydrophobins have only recently been structurally characterized through the first crystal structure determination of a protein of this class, Trichoderma reesei hydrophobin HFBII [Hakanpää, Paananen et al. (2004), J.,Biol.,Chem.279, 534,539]. The resolution of the HFBII structure has now been extended to an ultrahigh resolution of 0.75,Å. The structure was refined conventionally and multipole refinement has been initiated. The ultrahigh-resolution structure is analyzed here in detail and comparison is made to the previous atomic resolution structure of the same protein as well as to other ultrahigh-resolution structures found in the Protein Data Bank. [source]


    U1A RNA-binding domain at 1.8,Å resolution

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2003
    Peter B. Rupert
    The human U1A RNA-binding domain (RBD1) adopts one of the most common protein folds, the RNA-recognition motif, and is a paradigm for understanding RNA,protein interactions. A 2.8,Å resolution structure of the unbound RBD1 has previously been determined [Nagai et al. (1990). Nature (London), 348, 515,520] and revealed a well defined ,/, core with disordered termini. Using a longer construct, a 1.8,Å resolution structure of the unbound domain was determined that reveals an ordered C-terminal helix. The presence of this helix is consistent with a solution structure of the free domain [Avis et al. (1996). J. Mol. Biol.257, 398,411]; however, in the solution structure the helix occludes the RNA-binding surface. In the present structure, the helix occupies a position similar to that seen in a 1.9,Å resolution RNA,RBD1 complex structure [Oubridge et al. (1994). Nature (London), 372, 432,438]. The crystals in this study were grown from 2.2,M sodium malonate. It is possible that the high salt concentration helps to orient the C-terminal helix in the RNA-bound conformation by strengthening hydrophobic interactions between the buried face of the helix and the ,/, core of the protein. Alternatively, the malonate (several molecules of which are bound in the vicinity of the RNA-binding surface) may mimic RNA. [source]


    Atomic resolution structure of Escherichia coli dUTPase determined ab initio

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001
    A. González
    Cryocooled crystals of a mercury complex of Escherichia coli dUTPase diffract to atomic resolution. Data to 1.05,Å resolution were collected from a derivative crystal and the structure model was derived from a Fourier map with phases calculated from the coordinates of the Hg atom (one site per subunit of the trimeric enzyme) using the program ARP/wARP. After refinement with anisotropic temperature factors a highly accurate model of the bacterial dUTPase was obtained. Data to 1.45,Å from a native crystal were also collected and the 100,K structures were compared. Inspection of the refined models reveals that a large part of the dUTPase remains rather mobile upon freezing, with 14% of the main chain being totally disordered and with numerous side chains containing disordered atoms in multiple discrete conformations. A large number of those residues surround the active-site cavity. Two glycerol molecules (the cryosolvent) occupy the deoxyribose-binding site. Comparison between the native enzyme and the mercury complex shows that the active site is not adversely affected by the binding of mercury. An unexpected effect seems to be a stabilization of the crystal lattice by means of long-range interactions, making derivatization a potentially useful tool for further studies of inhibitor,substrate-analogue complexes of this protein at very high resolution. [source]


    The 1.30,Å resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2000
    Jane 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]


    Structure of a fibronectin type III-like module from Clostridium thermocellum

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010
    Markus Alahuhta
    The 1.6,Å resolution structure of a fibronectin type III-like module from Clostridium thermocellum (PDB code 3mpc) with two molecules in the asymmetric unit is reported. The crystals used for data collection belonged to space group P212121, with unit-cell parameters a = 35.43, b = 45.73, c = 107.72,Å, and the structure was refined to an R factor of 0.166. Structural comparisons found over 800 similar structures in the Protein Data Bank. The broad range of different proteins or protein domains with high structural similarity makes it especially demanding to classify these proteins. Previous studies of fibronectin type III-like modules have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules or as proteins that help large enzyme complexes remain soluble. [source]


    The 1.4,Å resolution structure of Paracoccus pantotrophus pseudoazurin

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
    Shabir Najmudin
    Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4,Å, with R and Rfree values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer,monomer recognition during dimer formation and for protein,protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins. [source]


    The 1.35,Å resolution structure of the phosphatase domain of the suppressor of T-cell receptor signaling protein in complex with sulfate

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
    Jean Jakoncic
    The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis,Menten constants at acidic pH. [source]


    The structure of PhaZ7 at atomic (1.2,Å) resolution reveals details of the active site and suggests a substrate-binding mode

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010
    Sachin Wakadkar
    Poly-(R)-hydroxyalkanoates (PHAs) are bacterial polyesters that are degraded by a group of enzymes known as PHA depolymerases. Paucimonas lemoignei PhaZ7 depolymerase is the only extracellular depolymerase that has been described as being active towards amorphous PHAs. A previously determined crystal structure of PhaZ7 revealed an ,/,-hydrolase fold and a Ser-His-Asp catalytic triad. In order to address questions regarding the catalytic mechanism and substrate binding, the atomic resolution structure of PhaZ7 was determined after cocrystallization with the protease inhibitor PMSF. The reported structure has the highest resolution (1.2,Å) of currently known depolymerase structures and shows a sulfur dioxide molecule covalently attached to the active-site residue Ser136. Structural comparison with the free PhaZ7 structure (1.45,Å resolution) revealed no major changes in the active site, suggesting a preformed catalytic triad. The oxyanion hole was found to be formed by the amide groups of Met137 and Asn49. Nine well ordered water molecules were located in the active site. Manual docking of a substrate trimer showed that the positions of these water molecules coincide well with the substrate atoms. It is proposed that these water molecules are displaced upon binding of the substrate. Furthermore, conformational changes were identified after comparison with a previously determined PhaZ7 dimer structure in a different space group. The changes were located in surface loops involved in dimer formation, indicating some flexibility of these loops and their possible involvement in polyester binding. [source]


    A micromolar O-sulfated thiohydroximate inhibitor bound to plant myrosinase

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010
    Arthur Besle
    The 1.6,Å resolution structure of the micromolar competitive inhibitor S -(N,N -dimethylaminoethyl) phenylacetothiohydroximate- O -sulfate bound to Sinapis alba myrosinase, a plant thioglucosidase, is reported. Myrosinase and its substrates, the glucosinolates, are part of the plant's defence system. The sulfate group and the phenyl group of the inhibitor bind to the aglycon-binding site of the enzyme, whereas the N,N -dimethyl group binds to the glucose-binding site and explains the large improvement in binding affinity compared with previous compounds. The structure suggests ways to increase the potency and specificity of the compound by improving the interactions with the hydrophobic pocket of the aglycon-binding site. [source]


    Comparison of GFL,GFR, complexes: further evidence relating GFL bend angle to RET signalling

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009
    Vimal Parkash
    Glial cell line-derived neurotrophic factor (GDNF) activates the receptor tyrosine kinase RET by binding to the GDNF-family receptor ,1 (GFR,1) and forming the GDNF2,GFR,12,RET2 heterohexamer complex. A previous crystal structure of the GDNF2,GFR,12 complex (PDB code 2v5e) suggested that differences in signalling in GDNF-family ligand (GFL) complexes might arise from differences in the bend angle between the two monomers in the GFL homodimer. Here, a 2.35,Å resolution structure of the GDNF2,GFR,12 complex crystallized with new cell dimensions is reported. The structure was refined to a final R factor of 22.5% (Rfree = 28%). The structures of both biological tetrameric complexes in the asymmetric unit are very similar to 2v5e and different from the artemin,GFR,3 structure, even though there is a small change in the structure of the GDNF. By comparison of all known GDNF and artemin structures, it is concluded that GDNF is more bent and more flexible than artemin and that this may be related to RET signalling. Comparisons also suggest that the differences between artemin and GDNF arise from the increased curvature of the artemin `fingers', which both increases the buried surface area in the monomer,monomer interface and changes the intermonomer bend angle. From sequence comparison, it is suggested that neuturin (the second GFL) adopts an artemin-like conformation, while persephin has a different conformation to the other three. [source]


    Application of statistical potentials to protein structure refinement from low resolution ab initio models

    BIOPOLYMERS, Issue 4 2003
    Hui Lu
    Abstract Recently ab initio protein structure prediction methods have advanced sufficiently so that they often assemble the correct low resolution structure of the protein. To enhance the speed of conformational search, many ab initio prediction programs adopt a reduced protein representation. However, for drug design purposes, better quality structures are probably needed. To achieve this refinement, it is natural to use a more detailed heavy atom representation. Here, as opposed to costly implicit or explicit solvent molecular dynamics simulations, knowledge-based heavy atom pair potentials were employed. By way of illustration, we tried to improve the quality of the predicted structures obtained from the ab initio prediction program TOUCHSTONE by three methods: local constraint refinement, reduced predicted tertiary contact refinement, and statistical pair potential guided molecular dynamics. Sixty-seven predicted structures from 30 small proteins (less than 150 residues in length) representing different structural classes (,, ,, ,,/,) were examined. In 33 cases, the root mean square deviation (RMSD) from native structures improved by more than 0.3 Å; in 19 cases, the improvement was more than 0.5 Å, and sometimes as large as 1 Å. In only seven (four) cases did the refinement procedure increase the RMSD by more than 0.3 (0.5) Å. For the remaining structures, the refinement procedures changed the structures by less than 0.3 Å. While modest, the performance of the current refinement methods is better than the published refinement results obtained using standard molecular dynamics. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 575,584, 2003 [source]


    Malonate-bound structure of the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ) and characterization of the native Fe2+ metal-ion preference

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2008
    Colin J. Jackson
    The structure of a malonate-bound form of the glycerophosphodiesterase from Enterobacter aerogenes, GpdQ, has been refined at a resolution of 2.2,Å to a final R factor of 17.1%. The structure was originally solved to 2.9,Å resolution using SAD phases from Zn2+ metal ions introduced into the active site of the apoenzyme [Jackson et al. (2007), J. Mol. Biol.367, 1047,1062]. However, the 2.9,Å resolution was insufficient to discern significant details of the architecture of the binuclear metal centre that constitutes the active site. Furthermore, kinetic analysis revealed that the enzyme lost a significant amount of activity in the presence of Zn2+, suggesting that it is unlikely to be a catalytically relevant metal ion. In this communication, a higher resolution structure of GpdQ is presented in which malonate is visibly coordinated in the active site and analysis of the native metal-ion preference is presented using atomic absorption spectroscopy and anomalous scattering. Catalytic implications of the structure and its Fe2+ metal-ion preference are discussed. [source]


    Atomic resolution structure of the double mutant (K53,56M) of bovine pancreatic phospholipase A2

    ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2006
    K. Sekar
    The structure of the double mutant K53,56M has previously been refined at 1.9,Å resolution using room-temperature data. The present paper reports the crystal structure of the same mutant K53,56M refined against 1.1,Å data collected using synchrotron radiation. A total of 116 main-chain atoms from 29 residues and 44 side chains are modelled in alternate conformations. Most of the interfacial binding residues are found to be disordered and alternate conformations could be recognized. The second calcium ion-binding site residue Glu92 adopts two alternate conformations. The minor and major conformations of Glu92 correspond to the second calcium ion bound and unbound states. [source]


    Structure-Based Calculation of Binding Affinities of ,2A -Adrenoceptor Agonists

    CHEMMEDCHEM, Issue 6 2007
    Balázs Balogh
    An atomic resolution structure of ,2a -adrenoceptor was constructed and 15 known agonists were docked into the optimized model and experimental binding free energies were estimated. The figure shows the binding of the agonist clonidine (sticks) to the core binding pocket of the adrenoceptor (blue cartoon, key residues are marked with sticks). [source]


    Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligand

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2010
    Laure Gabison
    Urate oxidase (uricase; EC 1.7.3.3; UOX) from Aspergillus flavus catalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cu etc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1,Å) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3,1.7,Å) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX,xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX,8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX,xanthine and the UOX,uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr,Lys,His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway. [source]


    A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009
    Artem Y. Lyubimov
    Cholesterol oxidase is a flavoenzyme that catalyzes the oxidation and isomerization of 3,-hydroxysteroids. Structural and mutagenesis studies have shown that Asn485 plays a key role in substrate oxidation. The side chain makes an NH..., interaction with the reduced form of the flavin cofactor. A N485D mutant was constructed to further test the role of the amide group in catalysis. The mutation resulted in a 1800-fold drop in the overall kcat. Atomic resolution structures were determined for both the N485L and N485D mutants. The structure of the N485D mutant enzyme (at 1.0,Å resolution) reveals significant perturbations in the active site. As predicted, Asp485 is oriented away from the flavin moiety, such that any stabilizing interaction with the reduced flavin is abolished. Met122 and Glu361 form unusual hydrogen bonds to the functional group of Asp485 and are displaced from the positions they occupy in the wild-type active site. The overall effect is to disrupt the stabilization of the reduced FAD cofactor during catalysis. Furthermore, a narrow transient channel that is shown to form when the wild-type Asn485 forms the NH..., interaction with FAD and that has been proposed to function as an access route of molecular oxygen, is not observed in either of the mutant structures, suggesting that the dynamics of the active site are altered. [source]


    Concanavalin A in a dimeric crystal form: revisiting structural accuracy and molecular flexibility

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2002
    Katherine A. Kantardjieff
    A structure of native concanavalin A (ConA), a hardy perennial of structural biology, has been determined in a dimeric crystal form at a resolution of 1.56,Å (space group C2221; unit-cell parameters a = 118.70, b = 101.38, c = 111.97,Å; two molecules in the asymmetric unit). The structure has been refined to an Rfree of 0.206 (R = 0.178) after iterative model building and phase-bias removal using Shake&wARP. Correspondence between calculated water,tyrosine interactions and experimentally observed structures near the saccharide-binding site suggests that the observed interactions between Tyr12 and water in various crystal forms are to be expected and are not unique to the presence of an active site. The present structure differs from previously reported atomic resolution structures of ConA in several regions and extends insight into the conformational flexibility of this molecule. Furthermore, this third, low-temperature, structure of ConA in a different crystal form, independently refined using powerful model-bias removal techniques, affords the opportunity to revisit assessment of accuracy and precision in high- or atomic resolution protein structures. It is illustrated that several precise structures of the same molecule can differ substantially in local detail and users of crystallographic models are reminded to consider the potential impact when interpreting structures. Suggestions on how to effectively represent ensembles of crystallographic models of a given molecule are provided. [source]