Gareth Chelvanayagam

Identification, characterization, and crystal structure of the Omega class glutathione transferases.

A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species andCaenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-Å resolution and has a characteristic GST fold (Protein Data Bank entry code 1eem). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.

Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site.

BACKGROUND Glutathione S-transferases (GSTs) comprise a multifunctional group of enzymes that play a critical role in the cellular detoxification process. These enzymes reduce the reactivity of toxic compounds by catalyzing their conjugation with glutathione. As a result of their role in detoxification, GSTs have been implicated in the development of cellular resistance to antibiotics, herbicides and clinical drugs and their study is therefore of much interest. In mammals, the cytosolic GSTs can be divided into five distinct classes termed alpha, mu, pi, sigma and theta. The human theta class GST, hGST T2-2, possesses several distinctive features compared to GSTs of other classes, including a long C-terminal extension and a specific sulfatase activity. It was hoped that the determination of the structure of hGST T2-2 may help us to understand more about this unusual class of enzymes. RESULTS Here we present the crystal structures of hGST T2-2 in the apo form and in complex with the substrates glutathione and 1-menaphthyl sulfate. The enzyme adopts the canonical GST fold with a 40-residue C-terminal extension comprising two helices connected by a long loop. The extension completely buries the substrate-binding pocket and occludes most of the glutathione-binding site. The enzyme has a purpose-built novel sulfate-binding site. The crystals were shown to be catalytically active: soaks with 1-menaphthyl sulfate result in the production of the glutathione conjugate and cleavage of the sulfate group. CONCLUSIONS hGST T2-2 shares less than 15% sequence identity with other GST classes, yet adopts a similar three-dimensional fold. The C-terminal extension that blocks the active site is not disordered in either the apo or complexed forms of the enzyme, but nevertheless catalysis occurs in the crystalline state. A narrow tunnel leading from the active site to the surface may provide a pathway for the entry of substrates and the release of products. The results suggest a molecular basis for the unique sulfatase activity of this GST.

Anatomy and evolution of proteins displaying the viral capsid jellyroll topology

In this paper the anatomy of 25 structures containing a jellyroll motif, consisting of eight antiparallel beta-strands forming a so-called beta-barrel, was investigated. This involved performing a careful structural alignment based on hydrogen bonds for the equivalent regions of the tertiary folds and a subsequent analysis of conserved amino acids, equivalenced residue-residue contacts, and various parameters describing the size, shape and other geometrical characteristics of these regions. It was found that the jellyroll motif is best viewed as a two-sheet wedge structure rather than a barrel. The more conserved parameters are discussed. A model of evolutionary development for the jellyroll fold in the various protein and viral structures is proposed.

An analysis of the helix-to-strand transition between peptides with identical sequence

An analysis of peptide segments with identical sequence but that differ significantly in structure was performed over non‐redundant databases of protein structures. We focus on those peptides, which fold into an α‐helix in one protein but a β‐strand in another. While the study shows that many such structurally ambivalent peptides contain amino acids with a strong helical preference collocated with amino acids with a strong strand preference, the results overwhelmingly indicate that the peptides environment ultimately dictates its structure. Furthermore, the first naturally occurring structurally ambivalent nonapeptide from evolutionary unrelated proteins is described, highlighting the intrinsic plasticity of peptide sequences. We even find seven proteins that show structural ambivalence under different conditions. Finally, a computer algorithm has been implemented to identify regions in a given sequence where secondary structure prediction programs are likely to make serious mispredictions. Proteins 2000;41:248–256.

A Roadmap for HLA-DR Peptide Binding Specificities

Peptide residue positional environments have previously been defined for class I MHC allelic products. These environments provide a less restrictive description of the traditional peptide binding pockets of class I molecules. When combined with the peptide anchor motifs that have been identified for some class I molecules, predictions as to likely motifs for other MHC molecules, which share the same potential environment can be made. Here, the same approach is used to derive peptide residue positional environments for class II MHC molecules. The environments are used to make predictions as to likely binding motifs for HLA-DR allelic products. The predictions are presented in the form of a Table and shown to have concordance with experimental results.

Prediction of protein folding pathways

Recent 1H nuclear magnetic resonance (n.m.r.) hydrogen exchange experiments on five different proteins have delineated the secondary structures formed in trapped, partially folded intermediates. The early forming structural elements are identifiable through a technique described in this work to predict folding pathways. The method assumes that the sequential selection of structural fragments such as alpha-helices and beta-strands involved in the folding process is founded upon the maximal burial of solvent accessible surface from both the formation of internal structure and substructure association. The substructural elements were defined objectively by major changes in main-chain direction. The predicted folding pathways are in complete correspondence with the n.m.r. results in that the formed structural fragments found in the folding intermediates are those predicted earliest in the pathways. The technique was also applied to proteins of known tertiary structure and with fold similar to one of the five proteins examined by 1H n.m.r. The pathways for these structures also showed general consistency with the n.m.r. observations, suggesting conservation of a secondary structural framework or molten globule about which folding nucleates and proceeds.

Proteolytic processing of peptides in the lumen of the endoplasmic reticulum for antigen presentation by major histocompatibility class I

We have tested the hypothesis that MHC class I molecules are actively involved as protease in the production of natural MHC class I ligands. First, the structure of a class I molecule was analyzed for homology with catalytic sites of known proteases. While several clusters of amino acids in the restriction element resembled protease active sites, structural discrepancies and the influence of nearby residues suggest that these sites are unlikely to have protease activity. Second, we have tested the presentation of viral cytotoxic T cell determinants with affinity for the same restriction element (H‐2Kd or Kk), when targeted as tandem peptides into the endoplasmic reticulum. Peptide transporter‐defective cells were used to exclude cleavage of the tandem peptides by cytosolic proteases. Cleavage by signal peptidase of the tandem peptides was ascertained. The C‐terminal peptides in the tandem arrays were almost exclusively presented, suggesting that an aminopeptidase in the endoplasmic reticulum degraded the N‐terminally positioned peptides. This result is inconsistent with an MHC class I‐catalyzed cleavage following binding of longer peptides in the cleft of the restriction elements. Finally, we conclusively show that an aminopeptidase in the endoplasmic reticulum is also involved in antigen presentation in cells with a functional peptide transporter.

Peptide binding motifs and specificities for HLA-DQ molecules

Abstract HLA-DQ molecules have been associated with susceptibility to a number of autoimmune and other diseases, possibly through the peptide repertoire that can be presented by different allelic products. It is thus of importance to understand which peptides can be bound by different HLA-DQ allelic products. Recently, a model for HLA-DQ has been described and used to derive peptide positional environments for HLA-DQ allelic products. By combining the peptide positional environments with known HLA-DQ peptide binding motifs, a set of predictions of likely anchor motifs for many of the products of HLA-DQ allelic variants are made and presented in a table referred to as a roadmap for HLA-DQ peptide binding specificities.

Polymorphism of phase II enzymes: identification of new enzymes and polymorphic variants by database analysis.

The Phase II enzymes of xenobiotic metabolism are characterized by a high level of substrate diversity and genetic polymorphism. Genetic polymorphism of the Phase II enzymes can be of substantial clinical significance as some variants have differences in substrate specificity, stability and levels of expression. Variation in these factors can give rise to abnormal drug metabolism and susceptibility to carcinogens and toxins. A new approach to the discovery of additional members of Phase II enzyme families and the identification of polymorphic variants using searches of the EST databases has been investigated. The examples provided demonstrate that relatively simple search strategies can be highly productive.

A homology model for the human theta‐class glutathione transferase T1–1

A manual threading approach is used to model the human glutathione transferase T1–1 based on the coordinates of the related Theta class enzyme T2–2. The low level of sequence identity (about 20%), found in the C‐terminal extension in conjunction with a relative deletion of about five residues makes this a challenging modeling problem. The C‐terminal extension contributes to the active site of the molecule and is thus of particular interest for understanding the molecular mechanism of the enzyme. Manual docking of known substrates and non‐substrates has implicated potential candidates for the T1–1 catalytic residues involved in the dehalogenation and epoxide‐ring opening activities. These include the conserved Theta class residues Arg 107, Trp 115, and the conserved GSTT1 subclass residue His 176. Also, the residue at position 234 is implicated in the modulation of T1–1 activity with different substrates between species. Proteins 33:444–454, 1998.