Alexander W. Schüttelkopf

PRODRG: a tool for high-throughput crystallography of protein-ligand complexes

The small-molecule topology generator PRODRG is described, which takes input from existing coordinates or various two-dimensional formats and automatically generates coordinates and molecular topologies suitable for X-ray refinement of protein-ligand complexes. Test results are described for automatic generation of topologies followed by energy minimization for a subset of compounds from the Cambridge Structural Database, which shows that, within the limits of the empirical GROMOS87 force field used, structures with good geometries are generated. X-ray refinement in X-PLOR/CNS, REFMAC and SHELX using PRODRG-generated topologies produces results comparable to refinement with topologies from the standard libraries. However, tests with distorted starting coordinates show that PRODRG topologies perform better, both in terms of ligand geometry and of crystallographic R factors.

Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc3 as a substrate (Km = 3.8 mM, kcat = 0.55 s-1), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanism-based inhibitors for this important class of enzymes.

Structural insights into mechanism and specificity of O ‐GlcNAc transferase

Post‐translational modification of protein serines/threonines with N‐acetylglucosamine (O‐GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O‐GlcNAcylation is regulated by O‐GlcNAc transferase (OGT) and O‐GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O‐GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X‐ray crystallography and mutagenesis, that OGT adopts the (metal‐independent) GT‐B fold and binds a UDP‐GlcNAc analogue at the bottom of a highly conserved putative peptide‐binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide‐binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.

O -GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.

Molecular Mechanisms of Yeast Cell Wall Glucan Remodeling

Yeast cell wall remodeling is controlled by the equilibrium between glycoside hydrolases, glycosyltransferases, and transglycosylases. Family 72 glycoside hydrolases (GH72) are ubiquitous in fungal organisms and are known to possess significant transglycosylase activity, producing elongated β(1–3) glucan chains. However, the molecular mechanisms that control the balance between hydrolysis and transglycosylation in these enzymes are not understood. Here we present the first crystal structure of a glucan transglycosylase, Saccharomyces cerevisiae Gas2 (ScGas2), revealing a multidomain fold, with a (βα)8 catalytic core and a separate glucan binding domain with an elongated, conserved glucan binding groove. Structures of ScGas2 complexes with different β-glucan substrate/product oligosaccharides provide “snapshots” of substrate binding and hydrolysis/transglycosylation giving the first insights into the mechanisms these enzymes employ to drive β(1–3) glucan elongation. Together with mutagenesis and analysis of reaction products, the structures suggest a “base occlusion” mechanism through which these enzymes protect the covalent protein-enzyme intermediate from a water nucleophile, thus controlling the balance between hydrolysis and transglycosylation and driving the elongation of β(1–3) glucan chains in the yeast cell wall.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence.

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.

Screening-based Discovery and Structural Dissection of a Novel Family 18 Chitinase Inhibitor *

Family 18 chitinases play key roles in the life cycles of a variety of organisms ranging from bacteria to man. Very recently it has been shown that one of the mammalian chitinases is highly overexpressed in the asthmatic lung and contributes to the pathogenic process through recruitment of inflammatory cells. Although several potent natural product chitinase inhibitors have been identified, their chemotherapeutic potential or their use as cell biological tools is limited due to their size, complex chemistry, and limited availability. We describe a virtual screening-based approach to identification of a novel, purine-based, chitinase inhibitor. This inhibitor acts in the low micromolar (Ki = 2.8 ± 0.2 μm) range in a competitive mode. Dissection of the binding mode by x-ray crystallography reveals that the compound, which consists of two linked caffeine moieties, binds in the active site through extensive and not previously observed stacking interactions with conserved, solvent exposed tryptophans. Such exposed aromatics are also present in the structures of many other carbohydrate processing enzymes. The compound exhibits favorable chemical properties and is likely to be useful as a general scaffold for development of pan-family 18 chitinase inhibitors.

Human OGA binds substrates in a conserved peptide recognition groove.

Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.

Structural basis of reduction-dependent activation of human cystatin F

Cystatins are important natural cysteine protease inhibitors targeting primarily papain-like cysteine proteases, including cathepsins and parasitic proteases like cruzipain, but also mammalian asparaginyl endopeptidase. Mammalian cystatin F, which is expressed almost exclusively in hematopoietic cells and accumulates in lysosome-like organelles, has been implicated in the regulation of antigen presentation and other immune processes. It is an unusual cystatin superfamily member with a redox-regulated activation mechanism and a restricted specificity profile. We describe the 2.1Å crystal structure of human cystatin F in its dimeric “off” state. The two monomers interact in a fashion not seen before for cystatins or cystatin-like proteins that is crucially dependent on an unusual intermolecular disulfide bridge, suggesting how reduction leads to monomer formation and activation. Strikingly, core sugars for one of the two N-linked glycosylation sites of cystatin F are well ordered, and their conformation and interactions with the protein indicate that this unique feature of cystatin F may modulate its inhibitory properties, in particular its reduced affinity toward asparaginyl endopeptidase compared with other cystatins.