David I. Stuart

ESPript: analysis of multiple sequence alignments in PostScript.

MOTIVATION The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

The atomic structure of the bluetongue virus core.

The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 Å. This transcriptionally active compartment, 700 Å in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.

Structural basis for the recognition of hydroxyproline in HIF-1|[alpha]| by pVHL

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1α is the oxygen-regulated subunit of HIF-1, an αβ heterodimeric complex. HIF-1α is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel–Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1α by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1α. The crystal structure of a hydroxylated HIF-1α peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

High resolution structures of HIV-1 RT from four RT-inhibitor complexes.

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 Å for all data to 2.2 Å. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/ conf igurational rearrangement of the compounds.

Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1

ABSTRACT Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognition is a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

The Interaction Properties of Costimulatory Molecules Revisited

B7-1 and B7-2 are generally thought to have comparable structures and affinities for their receptors, CD28 and CTLA-4, each of which is assumed to be bivalent. We show instead (1) that B7-2 binds the two receptors more weakly than B7-1, (2) that, relative to its CTLA-4 binding affinity, B7-2 binds CD28 2- to 3-fold more effectively than B7-1, (3) that, unlike B7-1, B7-2 does not self-associate, and (4) that, in contrast to CTLA-4 homodimers, which are bivalent, CD28 homodimers are monovalent. Our results indicate that B7-1 markedly favors CTLA-4 over CD28 engagement, whereas B7-2 exhibits much less bias. We propose that the distinct structures and binding properties of B7-1 and B7-2 account for their overlapping but distinct effects on T cell responses.

A functional and structural basis for TCR cross-reactivity in multiple sclerosis

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501–MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

A mechanism for initiating RNA-dependent RNA polymerization.

In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 Å resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage φ6 (refs 3, 4) is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.

Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 Å resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded β-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.

Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β2-microglobulin. A flexible loop of the α3 domain (residues 223–229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody–antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR–peptide–MHC interactions.