Christopher J. Squire

Crystal structures of F420-dependent glucose-6-phosphate dehydrogenase FGD1 involved in the activation of the anti-tuberculosis drug candidate PA-824 reveal the basis of coenzyme and substrate binding.

The modified flavin coenzyme F420 is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F420-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F420 for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F420 and citrate at resolutions of 1.90 and 1.95Å, respectively. The structure reveals a highly specific F420 binding mode, which is shared with several other F420-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F420 and is shown to be a competitive inhibitor (IC50 43 μm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F420 and helps promote a butterfly bend conformation that facilitates F420 reduction and catalysis.

Defining the Potassium Binding Region in an Apple Terpene Synthase

Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M2+) metal ion-assisted electrophilic attack. In addition to M2+, monovalent cations (M+) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple α-farnesene synthase (MdAFS1), which has a dependence upon potassium (K+), was used to identify active site regions that may be important for M+ binding. Protein homology modeling revealed a surface-exposed loop (H-αl loop) in MdAFS1 that fulfilled the necessary requirements for a K+ binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K+ response and strongly implicated specific residues in direct K+ binding. The role of the H-αl loop in terpene synthase K+ coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M+ binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K+ activation in other terpene synthases and establish a new role for the H-αl loop region in terpene synthase catalysis.

Structure and function of GlmU from Mycobacterium tuberculosis.

Antibiotic resistance is a major issue in the treatment of infectious diseases such as tuberculosis. Existing antibiotics target only a few cellular pathways and there is an urgent need for antibiotics that have novel molecular mechanisms. The glmU gene is essential in Mycobacterium tuberculosis, being required for optimal bacterial growth, and has been selected as a possible drug target for structural and functional investigation. GlmU is a bifunctional acetyltransferase/uridyltransferase that catalyses the formation of UDP-GlcNAc from GlcN-1-P. UDP-GlcNAc is a substrate for two important biosynthetic pathways: lipopolysaccharide and peptidoglycan synthesis. The crystal structure of M. tuberculosis GlmU has been determined in an unliganded form and in complex with GlcNAc-1-P or UDP-GlcNAc. The structures reveal the residues that are responsible for substrate binding. Enzyme activities were characterized by (1)H NMR and suggest that the presence of acetyl-coenzyme A has an inhibitory effect on uridyltransferase activity.

In Vitro Studies with Methylproamine A Potent New Radioprotector

New analogues of the minor groove binding ligand Hoechst 33342 have been investigated in an attempt to improve radioprotective activity. The synthesis, DNA binding, and in vitro radioprotective properties of methylproamine, the most potent derivative, are reported. Experiments with V79 cells have shown that methylproamine is ∼100-fold more potent than the classical aminothiol radioprotector WR1065. The crystal structures of methylproamine and proamine complexes with the dodecamer d(CGCGAATTCGCG)2 confirm that the new analogues also are minor groove binders. It is proposed that the DNA-bound methylproamine ligand acts as a reducing agent by an electron transfer mechanism, repairing transient radiation-induced oxidizing species on DNA.

The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)4 and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)4 quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions.

Crystal Structures of Three Classes of Non-Steroidal Anti-Inflammatory Drugs in Complex with Aldo-Keto Reductase 1C3

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Significance We describe an unprecedented type of intramolecular cross-link in a protein molecule, which we have found in the repetitive domains of a cell surface adhesin from the Gram-positive organism Clostridium perfringens. From high-resolution crystal structures of the protein, coupled with MS, we show that these domains contain intramolecular ester bonds joining Thr and Gln side chains. These bonds are generated autocatalytically by a serine protease-like mechanism and provide the long, thin protein with greatly enhanced mechanical strength and protection from proteolytic attack. The bonds provide an intriguing parallel with the internal isopeptide bonds that stabilize Gram-positive pili. Bioinformatics analysis suggests that these intramolecular ester bonds are widespread and common in cell surface adhesion proteins from Gram-positive bacteria. Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, attachment to host cells, and pathogenesis. These proteins, and protein assemblies, such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such cross-links can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins.

Synthesis and structure-activity relationships of soluble 8-substituted 4-(2-chlorophenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of the Wee1 and Chk1 checkpoint kinases.

Pyrrolo[3,4-c]carbazoles bearing solubilising basic side chains at the 8-position retain potent Wee1 and Chk1 inhibitory properties in isolated enzyme assays, and evidence of G2/M checkpoint abrogation in several cellular assays. Co-crystal structure studies confirm that the primary binding to the Wee1 enzyme is as described previously, with the C-8 side chains residing in an area of bulk tolerance.

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G2 and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

Poxviral Ankyrin Proteins

Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.