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Gary L Gilliland

Publications and source records attributed to Gary L Gilliland.

At least 19 recordsLinked to original sources
PubMed2006

Structural basis for sequential cleavage of fibrinopeptides upon fibrin assembly.

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.

Animals↗
PubMed2006

Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.

The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 +/- 0.05 mM and a k(cat) of 60 s(-1) per active site (at 37 degrees C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 A resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

Amino Acids, Aromatic↗
PubMed2005

Crystal structure of the bacterial YhcH protein indicates a role in sialic acid catabolism.

The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-A resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded beta-helix. Two antiparallel beta-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

Amino Acid Sequence↗
PubMed2004

Crystal structure of the YffB protein from Pseudomonas aeruginosa suggests a glutathione-dependent thiol reductase function.

BACKGROUND: The yffB (PA3664) gene of Pseudomonas aeruginosa encodes an uncharacterized protein of 13 kDa molecular weight with a marginal sequence similarity to arsenate reductase from Escherichia coli. The crystal structure determination of YffB was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. RESULTS: The structure was determined at 1.0 A resolution by single-wavelength anomalous diffraction. The fold is very similar to that of arsenate reductase, which is an extension of the thioredoxin fold. CONCLUSION: Given the conservation of the functionally important residues and the ability to bind glutathione, YffB is likely to function as a GSH-dependent thiol reductase.

Arsenite Transporting ATPases↗
PubMed2004

Crystal structure of the complex between thrombin and the central "E" region of fibrin.

Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized d-Phe-Pro-Arg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E(ht), corresponding to the central region of human fibrin, and solved its structure at 3.65-A resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E(ht) in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E(ht). Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A alpha chain Trp-33, Phe-35, Asp-38, Glu-39, the B beta chain Ala-68 and Asp-69, and the gamma chain Asp-27 and Ser-30 of E(ht) form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

Animals↗
PubMed2004

Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.

Amino Acid Substitution↗
PubMed2004

Ongoing development of two-dimensional polyacrylamide gel electrophoresis data standards.

We present an approach toward standardizing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) data in support of developing a globally relevant proteomics consensus in order to provide more efficient database querying and data comparisons through the establishment of the necessary definitions and interdisciplinary reference fields for both the 2-D PAGE community, particularly in the proteomics area, and the clinical and experimental biological research communities, in general. This article covers the need for unifying the 2-D PAGE data through a common data repository, and its usefulness in data standards and data interoperability.

Databases, Protein↗
PubMed2004

Data standards for proteomics: mitochondrial two-dimensional polyacrylamide gel electrophoresis data as a model system.

Proteomics has emerged as a major discipline that led to a re-examination of the need for consensus and a nationally sanctioned set of proteomics technology standards. Such standards for databases and data reporting may be applied to two-dimensional polyacrylamide gel electrophoresis (2D PAGE) technology as a pilot project for assessing global and national needs in proteomics, and the role of the National Institute of Standards and Technology (NIST) and other similar standards and measurement organizations. The experience of harmonizing the heterogeneous data included in the Protein Data Bank (PDB) provides a paradigm for technology in an area where significant heterogeneity in technical detail and data storage has evolved. Here we propose an approach toward standardizing mitochondrial 2D PAGE data in support of a globally relevant proteomics consensus.

Journal Article↗
PubMed2004

MitoMorphy: an alignment and annotation tool for human mitochondrial DNA polymorphisms.

MitoMorphy uses a number of publicly available human mitochondrial DNA (mtDNA) sequences from different ethnic groups to compare and annotate the associated polymorphic data. It provides an integrated display of mtDNA sequence comparison, sequence variation, and annotation for 695 different mtDNA sequences from many different ethnic groups around the world.

Journal Article↗
PubMed2004

Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism.

The ygfZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.

Amino Acid Sequence↗
PubMed2003

Conserved structural elements in glutathione transferase homologues encoded in the genome of Escherichia coli.

Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins. The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme. The enzyme consists of two domains. The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical. The majority of the consensus residues (12/17) reside in the N-terminal domain. Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains. The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain. The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids. The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively.

Amino Acid Sequence↗
PubMed2003

Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site.

BACKGROUND: The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function. RESULTS: The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid. CONCLUSION: The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

Binding Sites↗
PubMed2003

Crystal structure of the YajQ protein from Haemophilus influenzae reveals a tandem of RNP-like domains.

A hypothetical protein encoded by the gene YajQ of Haemophilus influenzae was selected, as part of a structural genomics project, for X-ray crystallographic structure determination and analysis to assist with the functional assignment. The protein is present in most bacteria, but not in archaea or eukaryotes. The amino acid sequence has no homology to that of other proteins. The YajQ protein was cloned, expressed, and the crystal structure determined at 2.1-A resolution by applying the multiwavelength anomalous dispersion method to a mercury derivative. The polypeptide chain is folded into two domains with identical folding topology. Each domain has a four-stranded antiparallel beta-sheet flanked on one side by two alpha-helices. This structural motif is a characteristic feature of many RNA-binding proteins. The tetrameric structure observed in the crystal suggests a possibility of binding two stretches of double-stranded nucleic acid.

Amino Acid Sequence↗
PubMed2003

Crystal structure of the YchF protein reveals binding sites for GTP and nucleic acid.

The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 A resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced alpha-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an alpha-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.

Amino Acid Sequence↗
PubMed2002

Local protein dynamics and catalysis: detection of segmental motion associated with rate-limiting product release by a glutathione transferase.

Glutathione transferase rGSTM1-1 catalyzes the addition of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, a reaction in which the chemical step is 60-fold faster than the physical step of product release. The hydroxyl group of Y115, located in the active site access channel, controls the egress of product from the active site. The Y115F mutant enzyme has a k(cat) (72 s(-)(1)) that is 3.6-fold larger than that of the native enzyme (20 s(-)(1)). Crystallographic observations and evidence from amide proton exchange kinetics are consistent with localized increases in the degree of segmental motion of the Y115F mutant that are coupled to the enhanced rate of product release. The loss of hydrogen bonding interactions involving the hydroxyl group of Y115 is reflected in subtle alterations in the backbone position, an increase in B-factors for structural elements that comprise the channel to the active site, and, most dramatically, a loss of well-defined electron density near the site of mutation. The kinetics of amide proton exchange are also enhanced by a factor between 3 and 12 in these regions, providing direct, quantitative evidence for changes in local protein dynamics affecting product release. The enhanced product release rate is proposed to derive from a small shift in the equilibrium population of protein conformers that permit egress of the product from the active site.

Amides↗