Biomedical subjects

David K Stammers

Publications and source records attributed to David K Stammers.

At least 19 recordsLinked to original sources

PubMed2006

Structure of vaccinia virus thymidine kinase in complex with dTTP: insights for drug design.

BACKGROUND: Development of countermeasures to bioterrorist threats such as those posed by the smallpox virus (variola), include vaccination and drug development. Selective activation of nucleoside analogues by virus-encoded thymidine (dThd) kinases (TK) represents one of the most successful strategies for antiviral chemotherapy as demonstrated for anti-herpes drugs. Vaccinia virus TK is a close orthologue of variola TK but also shares a relatively high sequence identity to human type 2 TK (hTK), thus achieving drug selectivity relative to the host enzyme is challenging. RESULTS: In order to identify any differences compared to hTK that may be exploitable in drug design, we have determined the crystal structure of VVTK, in complex with thymidine 5'-triphosphate (dTTP). Although most of the active site residues are conserved between hTK and VVTK, we observe a difference in conformation of residues Asp-43 and Arg-45. The equivalent residues in hTK hydrogen bond to dTTP, whereas in subunit D of VVTK, Asp-43 and Arg-45 adopt a different conformation preventing interaction with this nucleotide. Asp-43 and Arg-45 are present in a flexible loop, which is disordered in subunits A, B and C. The observed difference in conformation and flexibility may also explain the ability of VVTK to phosphorylate (South)-methanocarbathymine whereas, in contrast, no substrate activity with hTK is reported for this compound. CONCLUSION: The difference in conformation for Asp-43 and Arg-45 could thus be used in drug design to generate VVTK/Variola TK-selective nucleoside analogue substrates and/or inhibitors that have lower affinity for hTK.

Binding Sites↗

PubMed2006

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design.

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

Binding Sites↗

PubMed2006

Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32.

The import of nuclear-encoded proteins into chloroplasts is tightly controlled on both sides of the envelope membranes. Regulatory circuits include redox-control as well as calcium-regulation, with calmodulin being the likely mediator of the latter. Using affinity-chromatography on calmodulin-agarose, we could identify the inner envelope translocon component Tic32 as the predominant calmodulin-binding protein of this membrane. Calmodulin-binding assays corroborate the interaction for heterologously expressed as well as native Tic32. The interaction is calcium-dependent and is mediated by a calmodulin-binding domain between Leu-296 and Leu-314 close to the C-proximal end of the pea Tic32. We furthermore could establish Tic32 as a bona fide NADPH-dependent dehydrogenase. NADPH but not NADH or NADP(+) affects the interaction of Tic110 with Tic32 as well as Tic62. At the same time, dehydrogenase activity of Tic32 is affected by calmodulin. In particular, binding of NADPH and calmodulin to Tic32 appear to be mutually exclusive. These results suggest that redox modulation and calcium regulation of chloroplast protein import convene at the Tic translocon and that both could be mediated by Tic32.

Amino Acid Sequence↗

PubMed2006

Structure of Staphylococcus aureus guanylate monophosphate kinase.

Nucleotide monophosphate kinases (NMPKs) are potential antimicrobial drug targets owing to their role in supplying DNA and RNA precursors. The present work reports the crystal structure of Staphylococcus aureus guanylate monophosphate kinase (SaGMK) at 1.9 A resolution. The structure shows that unlike most GMKs SaGMK is dimeric, confirming the role of the extended C-terminus in dimer formation as first observed for Escherichia coli GMK (EcGMK). One of the two SaGMK dimers within the crystal asymmetric unit has two monomers in different conformations: an open form with a bound sulfate ion (mimicking the beta-phosphate of ATP) and a closed form with bound GMP and sulfate ion. GMP-induced domain movements in SaGMK can thus be defined by comparison of these conformational states. Like other GMKs, the binding of GMP firstly triggers a partial closure of the enzyme, diminishing the distance between the GMP-binding and ATP-binding sites. In addition, the closed structure shows the presence of a potassium ion in contact with the guanine ring of GMP. The potassium ion appears to form an integral part of the GMP-binding site, as the Tyr36 side chain has significantly moved to form a metal ion-ligand coordination involving the lone pair of the side-chain O atom. The potassium-binding site might also be exploited in the design of novel inhibitors.

Amino Acid Sequence↗

PubMed2006

Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine.

Aspartokinase III (AKIII) from Escherichia coli catalyzes an initial commitment step of the aspartate pathway, giving biosynthesis of certain amino acids including lysine. We report crystal structures of AKIII in the inactive T-state with bound feedback allosteric inhibitor lysine and in the R-state with aspartate and ADP. The structures reveal an unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat. Comparison of R- and T-state AKIII indicates that binding of lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a "latch", the beta15-alphaK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. Lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by lysine.

Allosteric Regulation↗

PubMed2006

Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5'-monophosphate.

The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5'-monophosphate (CMP) has been determined at 2.3 angstroms resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by approximately 16 degrees towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK-CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

Amino Acid Sequence↗

PubMed2006

Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 A resolution.

The P(II) signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. P(II)-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single P(II) protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the P(II) protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 A. Comparison of the structure with those of other P(II) proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria P(II) protein shares functions with GlnB/GlnK of enteric bacteria.

Bacterial Proteins↗

PubMed2006

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation.

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).

Antiviral Agents↗

PubMed2006

Mutations distal to the substrate site can affect varicella zoster virus thymidine kinase activity: implications for drug design.

Varicella zoster virus encodes a thymidine kinase responsible for the activation of antiherpetic nucleoside prodrugs such as acyclovir. In addition, herpes virus thymidine kinases are being explored in gene/chemotherapy strategies aimed at developing novel antitumor therapies. To investigate and improve compound selectivity, we report here structure-based site-directed mutagenesis studies of varicella zoster virus thymidine kinase (VZVTK). Earlier reports showed that mutating residues at the core of the VZVTK active site invariably destroyed activity; hence, we targeted more distal residues. Based on the VZVTK crystal structure, we constructed six mutants (E59S, R84V, H97Y/A, and Y21H/E) and tested substrate activity and competitive inhibition for several compound series. All VZVTK mutants tested retained significant phosphorylation activity with dThd as substrate, apart from Y21E (350-fold diminution in the k(cat)/K(m)). Some mutations give slightly improved affinities: bicyclic nucleoside analogs (BCNAs) with a p-alkyl-substituted phenyl group seem to require aromatic ring stacking interactions with residue 97 for optimal inhibitory effect. Mutation Y21E decreased the IC(50) value for the BCNA 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (Cf1368) 4-fold, whereas mutation Y21H increased the IC(50) value by more than 15-fold. These results suggest that residue 21 is important for BCNA selectivity and might explain why HSV1TK is unable to bind BCNAs. Other mutants, such as the E59S and R84V thymidine kinases, which in wild-type VZVTK stabilize the dimer interface, give opposite results regarding the level of sensitivity to BCNAs. The work described here shows that distal mutations that affect the VZVTK active-site may help in the design of more selective substrates for gene suicide therapy or as anti-varicella zoster virus drugs.

Antiviral Agents↗

PubMed2006

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix alpha3 shifting position initially, followed by movement of alpha2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.

Amino Acid Sequence↗

PubMed2006

Structure-activity relationship studies of novel benzophenones leading to the discovery of a potent, next generation HIV nonnucleoside reverse transcriptase inhibitor.

Despite the progress of the past two decades, there is still considerable need for safe, efficacious drugs that target human immunodeficiency virus (HIV). This is particularly true for the growing number of patients infected with virus resistant to currently approved HIV drugs. Our high throughput screening effort identified a benzophenone template as a potential nonnucleoside reverse transcriptase inhibitor (NNRTI). This manuscript describes our extensive exploration of the benzophenone structure-activity relationships, which culminated in the identification of several compounds with very potent inhibition of both wild type and clinically relevant NNRTI-resistant mutant strains of HIV. These potent inhibitors include 70h (GW678248), which has in vitro antiviral assay IC(50) values of 0.5 nM against wild-type HIV, 1 nM against the K103N mutant associated with clinical resistance to efavirenz, and 0.7 nM against the Y181C mutant associated with clinical resistance to nevirapine. Compound 70h has also demonstrated relatively low clearance in intravenous pharmacokinetic studies in three species, and it is the active component of a drug candidate which has progressed to phase 2 clinical studies.

Alkynes↗

PubMed2006

Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138.

Lys101Glu is a drug resistance mutation in reverse transcriptase clinically observed in HIV-1 from infected patients treated with the non-nucleoside inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the surface of the NNRTI pocket where it interacts across the reverse transcriptase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine contacts Lys101 and Glu138 only indirectly, via water molecules, thus the structural basis of drug resistance induced by Lys101Glu is unclear. We have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu) in complexes with nevirapine to 2.5 A, allowing the determination of water structure within the NNRTI-binding pocket, essential for an understanding of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have remarkably similar protein conformations to wild-type RT, except for significant movement of the mutated side-chains away from the NNRTI pocket induced by charge inversion. There are also small shifts in the position of nevirapine for both mutant structures which may influence ring stacking interactions with Tyr181. However, the reduction in hydrogen bonds in the drug-water-side-chain network resulting from the mutated side-chain movement appears to be the most significant contribution to nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away from the NNRTI pocket can also explain the resistance of RT(Lys101Glu) to efavirenz but in this case is due to a loss of side-chain contacts with the drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in that it can give rise to both direct and indirect mechanisms of drug resistance, which are inhibitor-dependent.

Anti-HIV Agents↗

PubMed2005

Molecular architecture and ligand recognition determinants for T4 RNA ligase.

RNA ligase type 1 from bacteriophage T4 (Rnl1) is involved in countering a host defense mechanism by repairing 5'-PO4 and 3'-OH groups in tRNA(Lys). Rnl1 is widely used as a reagent in molecular biology. Although many structures for DNA ligases are available, only fragments of RNA ligases such as Rnl2 are known. We report the first crystal structure of a complete RNA ligase, Rnl1, in complex with adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPcPP). The N-terminal domain is related to the equivalent region of DNA ligases and Rnl2 and binds AMPcPP but with further interactions from the additional N-terminal 70 amino acids in Rnl1 (via Tyr37 and Arg54) and the C-terminal domain (Gly269 and Asp272). The active site contains two metal ions, consistent with the two-magnesium ion catalytic mechanism. The C-terminal domain represents a new all alpha-helical fold and has a charge distribution and architecture for helix-nucleic acid groove interaction compatible with tRNA binding.

Bacteriophage T4↗

PubMed2005

GTP cyclohydrolase II structure and mechanism.

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Binding Sites↗

PubMed2005

Crystal structure of nitrogen regulatory protein IIANtr from Neisseria meningitidis.

BACKGROUND: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. RESULTS: The NM-IIANtr was over-expressed in E. coli and was shown to be partially mono-phosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 A resolution. The structure of NM-IIANtr was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIAntr [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIANtr from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIAmtl complexed with HPr [PDB: 1J6T] indicates that NM-IIANtr binds in a similar way to the HPr-like enzyme in Neisseria. CONCLUSION: The structure of NM-IIANtr confirms its assignment as a homologue of the IIANtr proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIANtr protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism.

Arginine↗

PubMed2005

A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Animals↗

PubMed2005

Sample preparation and mass-spectrometric characterization of crystal-derived protein samples.

Mass spectrometry is often used to ascertain the accurate mass of purified protein samples prior to crystallization screening. However, in many cases data regarding the form of the protein crystallizing can also be useful, as this may differ from the original sample. Development of a simple method for the preparation and mass spectrometry of crystal-derived protein samples is described. The method is exemplified by the determination of the phosphorylation state of protein in a crystal derived from a mixture of phosphorylated and unphosphorylated protein.

Chromatography, High Pressure Liquid↗

PubMed2005

Crystal structure of CC3 (TIP30): implications for its role as a tumor suppressor.

CC3 (TIP30) is a protein with pro-apoptotic and anti-metastatic properties. The tumor suppressor effect of CC3 has been suggested to result from inhibition of nuclear transport by binding to importin betas or by regulating transcription through interaction in a complex with co-activator independent of AF-2 function (CIA) and the c-myc gene. Previous biochemical studies indicated that CC3 has protein kinase activity, and a structural similarity to cAMP-dependent protein kinase catalytic subunit was proposed. By contrast, bioinformatics studies suggested a relationship of CC3 to the short chain dehydrogenase reductase family. To clarify details of the CC3 structural family and ligand binding properties, we have determined the crystal structure of CC3 at 1.7-A resolution. CC3 has a short chain dehydrogenase reductase fold and binding specificity for NADPH, yet it is unlikely to be normally enzymatically active because it is monomeric. These structural results, in conjunction with data from earlier mutagenesis work on the nucleotide binding motif, suggest that NADPH binding is important for the biological activity of CC3, including interaction with importins and with the CIA/c-myc system. CC3 provides an example of the adaptation of a metabolic enzyme fold to include a regulatory role, as also seen in the case of the NADH-binding co-repressor CtBP.

Acetyltransferases↗