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PubMed1998

Crystal structure of the catalytic domain of protein-tyrosine phosphatase SHP-1.

The crystal structures of the protein-tyrosine phosphatase SHP-1 catalytic domain and the complex it forms with the substrate analogue tungstate have been determined and refined to crystallographic R values of 0.209 at 2.5 A resolution and 0.207 at 2.8 A resolution, respectively. Despite low sequence similarity, the catalytic domain of SHP-1 shows high similarity in secondary and tertiary structures with other protein-tyrosine phosphatases (PTPs). In contrast to the conformational changes observed in the crystal structures of PTP1B and Yersinia PTP, the WPD loop (Trp419-Pro428) in the catalytic domain of SHP-1 moves away from the substrate binding pocket after binding the tungstate ion. Sequence alignment and structural analysis suggest that the residues in the WPD loop, especially the amino acid following Asp421, are critical for the movement of WPD loop on binding substrates and the specific activity of protein-tyrosine phosphatases. Our mutagenesis and kinetic measurements have supported this hypothesis.

Amino Acid Sequence↗
PubMed2006

Investigating human P450s involved in drug metabolism via homology with high-resolution P450 crystal structures of the CYP2C subfamily.

The important role of high-resolution crystal structures of cytochrome P450 (CYP) enzymes for the generation of P450 models by homology is discussed. The main focus is on human P450 enzymes involved in drug metabolism, where the role of homology modelling has been emphasized in the recent literature. Report of the first human P450 crystal structure has provided an opportunity for comparison between those modelled from other crystallographic templates, and the recent substrate-bound rabbit CYP2C5 structure exemplifies the relevance of high-resolution template structures to generating 3-D models of P450s where the homology is relatively high. In particular, the homology models of human CYP1 and CYP2 family enzymes are presented, where good agreement with experiment findings are apparent.

Animals↗
PubMed2002

Synthesis and characterization of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine: crystal structures of 4,6-O-butylidene-alpha-D-glucopyranose, 4,6-O-butylidene-beta-D-glucopyranosylamine and 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine.

4,6-O-Butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. 1H and 13C NMR studies showed the presence of the beta-anomer, which has also been confirmed by the crystal structure. The molecular structure of this compound showed the presence of the tridentate ONO ligation-core. Both precursors, 4,6-O-butylidene-alpha-D-glucopyranose and 4,6-O-butylidene-beta-D-glucopyranosylamine were characterized using single crystal X-ray diffraction. The alpha-anomeric nature of the former and beta-anomeric nature of the latter were proposed based on 1H NMR studies and were confirmed by determining the crystal structures. In addition, the crystal structure of 4,6-O-butylidene-beta-D-glucopyranosylamine revealed the C-1-N-glycosylation. In all the three molecules, the saccharide unit exhibits a 4C(1) chair conformation. In the lattice, the molecules are connected by hydrogen-bond interactions. The conformation of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine is stabilized via an O-H...N intramolecular interaction, and each molecule in the lattice interacts with three neighboring molecules through hydrogen bonds of the type O-H...O and C-H...O.

Carbohydrate Conformation↗
PubMed2002

The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii.

The crystal structure of pyrrolidone-carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography. PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides. The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus furiosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefaciens. The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP. The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors.

Amino Acid Sequence↗
PubMed2005

Electronic structure of thiazine-indigo pigment on the basis of the crystal structure.

Thiazine-indigos are novel hydrogen-bonded pigments developed recently by Clariant that exhibit a variety of shades from yellow to red. Among these, a commercially available chlorinated derivative (THI) gives a color of vivid red, although it is only pale yellow in solution. For this reason, electronic characterization of THI has been carried out on the basis of the crystal structure. There are chains of intermolecular hydrogen bonds between the N-H group of one molecule and the O atom of the neighboring one along the b-axis, forming a two-dimensional hydrogen bond network. The direction of the transition dipole points along the long molecular axis, and the transition dipoles are arranged in "head-to-tail" fashion along the (b,-c) diagonal direction. The large bathochromic shift upon crystallization can be mostly attributed to excitonic interactions between transition dipoles arranged diagonally along the stacking axis as well as those in head-to-tail fashion on the molecular plane.

Journal Article↗
PubMed2001

The crystal structure of an aldehyde reductase Y50F mutant-NADP complex and its implications for substrate binding.

In order to understand more fully the structural features of aldo-keto reductases (AKRs) that determine their substrate specificities it would be desirable to obtain crystal structures of an AKR with a substrate at the active site. Unfortunately the reaction mechanism does not allow a binary complex between enzyme and substrate and to date ternary complexes of enzyme, NADP(H) and substrate or product have not been achieved. Previous crystal structures, in conjunction with numerous kinetic and theoretical analyses, have led to the general acceptance of the active site tyrosine as the general acid-base catalytic residue in the enzyme. This view is supported by the generation of an enzymatically inactive site-directed mutant (tyrosine-48 to phenylalanine) in human aldose reductase [AKR1B1]. However, crystallization of this mutant was unsuccessful. We have attempted to generate a trapped cofactor/substrate complex in pig aldehyde reductase [AKR1A2] using a tyrosine 50 to phenylalanine site-directed mutant. We have been successful in the generation of the first high resolution binary AKR-Y50F:NADP(H) crystal structure, but we were unable to generate any ternary complexes. The binary complex was refined to 2.2A and shows a clear lack of density due to the missing hydroxyl group. Other residues in the active site are not significantly perturbed when compared to other available reductase structures. The mutant binds cofactor (both oxidized and reduced) more tightly but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone as determined by fluorescence titrations. Attempts at substrate addition to the active site, either by cocrystallization or by soaking, were all unsuccessful using pyridine-3-aldehyde, 4-carboxybenzaldehyde, succinic semialdehyde, methylglyoxal, and other substrates. The lack of ternary complex formation, combined with the significant differences in the binding of barbitone provides some experimental proof of the proposal that the hydroxyl group on the active site tyrosine is essential for substrate binding in addition to its major role in catalysis. We propose that the initial event in catalysis is the binding of the oxygen moiety of the carbonyl-group of the substrate through hydrogen bonding to the tyrosine hydroxyl group.

Aldehyde Reductase↗
PubMed1996

Crystal structures of the recombinant kringle 1 domain of human plasminogen in complexes with the ligands epsilon-aminocaproic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic Acid.

The X-ray crystal structures of the complexes of the recombinant kringle 1 domain of human plasminogen (Klpg) with the ligands epsilon-aminocaproic acid (EACA) and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), which are representative of a class of in vivo antifibrinolytic agents, have been determined at 2.1 angstroms resolution. Each Klpg/ligand unit cell contained a dimer of the complexes, and some small differences were noted in the kringle/ligand interatomic distances within the monomeric components of the dimers. The structures obtained allowed predictions to be made of the amino acid residues of Klpg that are likely important to ligand binding. In the crystal structure, the anionic center of Klpg responsible for coordinating the amino group of the ligands is composed of both Asp54 and Asp56, and the cationic center that stabilizes binding of the carboxylate moiety of the ligands is Arg70, with a possible contribution from Arg34. A hydrogen bond between the carboxylate of the ligand to the hydroxyl group of Tyr63 also appears to contribute to the kringle/ligand binding energies. The methylene groups of the ligand are stablized in the binding pocket by van der Waals contacts with side-chain atoms of Trp61 and Tyr71. These conclusions are in general agreement with site-directed mutagenesis results that implicate many of the same amino acid residues in the binding process, thus showing that the crystal and solution structures are in basic accord with each other. Further comparisons of the X-ray crystal structures of the Klpg/ligand complexes with each other and with apo-Klpg show that while small differences in Klpg side-chain geometries may exist in the three structures, the binding pocket can be considered to be preformed in the apokringle and not substantially altered by the nature of the omega-amino acid ligand that is inserted into the site. From the similar geometries of the binding of EACA and AMCHA, it appears that the kon is an important component to the tighter binding of AMCHA to Klpg, as compared to EACA. Ordered solvation effects of the bound AMCHA may contribute to its longer lifetime on Klpg, thereby retarding koff, both effects thus accounting for the higher binding energy of AMCHA as compared to EACA.

Aminocaproic Acid↗
PubMed1998

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase.

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-A resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Omega loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Adenosine Triphosphate↗
PubMed2005

Crystal structure of the N-terminal domain of E. coli Lon protease.

We report here the first crystal structure of the N-terminal domain of an A-type Lon protease. Lon proteases are ubiquitous, multidomain, ATP-dependent enzymes with both highly specific and non-specific protein binding, unfolding, and degrading activities. We expressed and purified a stable, monomeric 119-amino acid N-terminal subdomain of the Escherichia coli A-type Lon protease and determined its crystal structure at 2.03 A (Protein Data Bank [PDB] code 2ANE). The structure was solved in two crystal forms, yielding 14 independent views. The domain exhibits a unique fold consisting primarily of three twisted beta-sheets and a single long alpha-helix. Analysis of recent PDB depositions identified a similar fold in BPP1347 (PDB code 1ZBO), a 203-amino acid protein of unknown function from Bordetella parapertussis, crystallized as part of a structural genomics effort. BPP1347 shares sequence homology with Lon N-domains and with a family of other independently expressed proteins of unknown functions. We postulate that, as is the case in Lon proteases, this structural domain represents a general protein and polypeptide interaction domain.

Amino Acid Sequence↗
PubMed1997

The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates.

The crystal structure of the recombinant form of rat liver fatty acid-binding protein was completed to 2.3 A and refined to an R factor of 19.0%. The structural solution was obtained by molecular replacement using superimposed polyalanine coordinates of six intracellular lipid-binding proteins as a search probe. The entire amino acid sequence of rat liver fatty acid-binding protein along with an amino-terminal formyl-methionine was modeled in the crystal structure. In addition, the crystal was obtained in the presence of oleic acid, and the initial electron density clearly showed two fatty acid molecules bound within a central cavity. The carboxylate of one fatty acid molecule interacts with arginine 122 and is shielded from free solvent. It has an overall bent conformation. The more solvent-exposed carboxylate of the other oleate is located near the helix-turn-helix that caps one end of the beta-barrel, while the acyl chain lies in the interior. The cavity contains both polar and nonpolar residues but also shows extensive hydrophobic character around the nonpolar atoms of the ligands. The primary and secondary oleate binding sites appear to be totally interdependent, mainly because favorable hydrophobic interactions form between both aliphatic chains.

Animals↗
PubMed2004

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides.

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.

Antineoplastic Agents↗
PubMed2000

Exploring the role of glutamine 50 in the homeodomain-DNA interface: crystal structure of engrailed (Gln50 --> ala) complex at 2.0 A.

We have determined the crystal structure of a complex containing the engrailed homeodomain Gln50 --> Ala variant (QA50) bound to the wild-type optimal DNA site (TAATTA) at 2.0 A resolution. Biochemical and genetic studies by other groups have suggested that residue 50 is an important determinant of differential DNA-binding specificity among homeodomains (distinguishing among various sites of the general form TAATNN). However, biochemical studies of the QA50 variant had revealed that it binds almost as tightly as the wild-type protein and with only modest changes in specificity. We have now determined the crystal structure of the QA50 variant to help understand the role of residue 50 in site-specific recognition. Our cocrystal structure shows some interesting changes in the water structure at the site of the substitution and shows some changes in the conformations of neighboring side chains. However, the structure, like the QA50 biochemical data, suggests that Gln50 plays a relatively modest role in determining the affinity and specificity of the engrailed homeodomain.

Crystallography↗
PubMed2006

Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme.

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.

Amino Acid Substitution↗
PubMed1994

The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution.

The crystal structure of manganese peroxidase (MnP) from the lignin-degrading basidiomycetous fungus Phanerochaete chrysosporium has been solved using molecular replacement techniques and refined to R = 0.20 at 2.0 A. The overall structure is similar to that of two other fungal peroxidases, lignin peroxidase from P. chrysosporium and Arthromyces ramosus peroxidase. Like the other fungal peroxidases, MnP has two structural calcium ions. MnP also has two N-acetylglucosamine residues N-linked to Asn131 that are readily visible in the electron density map. The active site, consisting of a proximal His ligand H-bonded to an Asp residue and a distal side peroxide binding pocket consisting of a catalytic His and Arg, is the same as in the aforementioned fungal peroxidases as well as yeast cytochrome c peroxidase. MnP differs in having five rather than four disulfide bonds. The additional disulfide bond, Cys341-Cys348, is located near the C terminus of the polypeptide chain. Importantly, a new cation binding site, which we propose is the manganese-binding site of MnP, was located in the crystal structure. The ligands constituting the Mn(2+)-binding site include Asp179, Glu35, Glu39, a heme propionate, and two water molecules. Electron transfer from Mn2+ to the heme edge or iron center is envisioned to occur through a sigma-bonded pathway along a heme propionate.

Amino Acid Sequence↗
PubMed1993

Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2'GMP.

RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.

Amino Acid Sequence↗
PubMed1992

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2003

Crystal structures of reversible ketone-Based inhibitors of the cysteine protease cruzain.

The crystal structures of two hydroxymethyl ketone inhibitors complexed to the cysteine protease cruzain have been determined at 1.1 and 1.2 A resolution, respectively. These high resolution crystal structures provide the first structures of non-covalent inhibitors bound to cruzain. A series of compounds were prepared and tested based upon the structures providing further insight into the key binding interactions.

Animals↗
PubMed1995

Small-molecule crystal structures as a structural basis for drug design.

For a long time, the crystal structures of small molecules were regarded as useful only for establishing the stereochemical formulae of the crystallized compounds. Recently, chemists have realized that in the study of the environment in the solid state there exists valuable structural information on the binding characteristics of chemical groups. Numerous comparisons have been made which show the nearly perfect correlation between small-molecule structural results, and the observed binding in receptor-substrate complexes. Moreover, the observed conformations of flexible substrates interacting with the neighbouring molecules in their crystal structures, can lead to valuable hypotheses on their conformation when bound in the active site of a biological macromolecule.

Journal Article↗