Magnus S. Alphey

The oxidoreductase ERp57 efficiently reduces partially folded in preference to fully folded MHC class I molecules

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone‐assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disulfides prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 specifically reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide‐loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N‐terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation.

Biosynthesis of isoprenoids: Crystal structure of 4-diphosphocytidyl-2C-methyl-d-erythritol kinase

4-Diphosphocytidyl-2C-methyl-d-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate. The 2-Å resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the α/β fold characteristic of the galactose kinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents.

High Resolution Crystal Structures of Siglec-7 INSIGHTS INTO LIGAND SPECIFICITY IN THE SIGLEC FAMILY

Sialic acid-binding immunoglobulin-like lectins (Siglecs) recognize sialylated glycoconjugates and play a role in cell-cell recognition. Siglec-7 is expressed on natural killer cells and displays unique ligand binding properties different from other members of the Siglec family. Here we describe the high resolution structures of the N-terminal V-set Ig-like domain of Siglec-7 in two crystal forms, at 1.75 and 1.9 Å. The latter crystal form reveals the full structure of this domain and allows us to speculate on the differential ligand binding properties displayed by members of the Siglec family. A fully ordered N-linked glycan is observed, tethered by tight interactions with symmetry-related protein molecules in the crystal. Comparison of the structure with that of sialoadhesin and a model of Siglec-9 shows that the unique preference of Siglec-7 for α(2,8)-linked disialic acid is likely to reside in the C-C′ loop, which is variable in the Siglec family. In the Siglec-7 structure, the ligand-binding pocket is occupied by a loop of a symmetry-related molecule, mimicking the interactions with sialic acid.

Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR–ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

The Synthesis of UDP-N-acetylglucosamine Is Essential for Bloodstream Form Trypanosoma brucei in Vitro and in Vivo and UDP-N-acetylglucosamine Starvation Reveals a Hierarchy in Parasite Protein Glycosylation

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

Structure of the macrocycle thiostrepton solved using the anomalous dispersion contribution of sulfur

The structure of a tetragonal crystal form of thiostrepton has been solved using the anomalous dispersive effects of five S atoms from high-redundancy data collected to 1.33 A resolution at the Cu Kalpha wavelength. Data measured to 1.02 A resolution with a synchrotron source were used for refinement. Details of the molecular structure, intramolecular and intermolecular interactions are given.

The identification of isoprenoids that bind in the intersubunit cavity of Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase by complementary biophysical methods.

The discovery of a distinct metabolic pathway, the non-mevalonate or 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for isoprenoid precursor biosynthesis, in eubacteria and apicomplexan parasites has revealed a new set of potential drug targets. The emphasis of research on this pathway has been on delineating the intermediates and the biochemical and structural characterization of component enzymes. Two new monoclinic crystal forms of recombinant Escherichia coli 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECP) synthase cocrystallized with (i) CMP and (ii) CMP and MECP show well defined electron density at the subunit interface suggestive of an isoprenoid-like ligand. 31P NMR analysis of the recombinant protein sample indicates the presence of bound diphosphate species and electrospray mass spectrometry identifies a mixture of isopentenyl diphosphate (and/or dimethylallyl diphosphate), geranyl diphosphate and farnesyl diphosphate in an approximate ratio of 1:4:2. The most prevalent species, geranyl diphosphate, was successfully modelled into the electron density, revealing the important protein-ligand interactions that stabilize binding of the isoprenoid. The observation that MECP synthase binds three metabolites that are produced by enzymes two, three and four stages downstream in isoprenoid biosynthesis suggests that feedback regulation of the non-mevalonate pathway is possible.

Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase - ligand recognition in a template for antimicrobial drug discovery.

4‐Diphosphocytidyl‐2C‐methyl‐d‐erythritol kinase (IspE) catalyses the ATP‐dependent conversion of 4‐diphosphocytidyl‐2C‐methyl‐d‐erythritol (CDPME) to 4‐diphosphocytidyl‐2C‐methyl‐d‐erythritol 2‐phosphate with the release of ADP. This reaction occurs in the non‐mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti‐infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4‐fluoro‐3,5‐dihydroxy‐4‐methylpent‐1‐enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP α‐phosphate not the binding site for the methyl‐d‐erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic α/β galacto‐homoserine‐mevalonate‐phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP‐ and CDPME‐binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well‐ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure‐based design of broad‐spectrum antimicrobial agents.

Synthesis and Characterization of Cytidine Derivatives that Inhibit the Kinase IspE of the Non-Mevalonate Pathway for Isoprenoid Biosynthesis

The enzymes of the non‐mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water‐soluble, cytidine‐based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co‐crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co‐crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well‐conserved ATP‐binding Gly‐rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure‐based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.

Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases

TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.