Biomedical subjects

Jingshan Ren

Publications and source records attributed to Jingshan Ren.

18 recordsLinked to original sources

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

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

Lysine methylation as a routine rescue strategy for protein crystallization.

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Bacillus anthracis↗

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

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↗

PubMed2005

HIV reverse transcriptase structures: designing new inhibitors and understanding mechanisms of drug resistance.

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. The selection of drug-resistant HIV is a key problem in the continued treatment of the infection and thus new drugs are required. A significant body of information consisting of HIV-1 RT crystal structures with bound inhibitors has become available during the past several years, and, increasingly, such data will be of use in developing novel inhibitors. Two examples of crystal structures of HIV-1 RT with bound inhibitors have been published recently, one with the non-nucleoside CP94707 and the second with the nucleotide analogue drug tenofovir. Such structures will help the design of new drugs and improve our understanding of the mechanisms of resistance.

Drug Design↗

PubMed2004

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 1.

We have used a structure-based approach to design a novel series of non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Detailed analysis of a wide range of crystal structures of HIV-1 RT-NNRTI complexes together with data on drug resistance mutations has identified factors important for tight binding of inhibitors and resilience to mutations. Using this approach we have designed and synthesized a novel series of quinolone NNRTIs. Crystal structure analysis of four of these compounds in complexes with HIV-1 RT confirms the predicted binding modes. Members of this quinolone series retain high activity against the important resistance mutations in RT at Tyr181Cys and Leu100Ile.

Anti-HIV Agents↗

PubMed2004

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 2.

HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) are part of the combination therapy currently used to treat HIV infection. The features of a new NNRTI drug for HIV treatment must include selective potent activity against both wild-type virus as well as against mutant virus that have been selected by use of current antiretroviral treatment regimens. Based on analogy with known HIV-1 NNRTI inhibitors and modeling studies utilizing the X-ray crystal structure of inhibitors bound in the HIV-1 RT, a series of substituted 2-quinolones was synthesized and evaluated as HIV-1 inhibitors.

Alkynes↗

PubMed2004

Modulation of the ligand binding properties of the transcription repressor NmrA by GATA-containing DNA and site-directed mutagenesis.

NmrA is a negative transcription-regulating protein that binds to the C-terminal region of the GATA transcription-activating protein AreA. The proposed molecular mechanism of action for NmrA is to inhibit AreA binding to its target promoters. In contrast to this proposal, we report that a C-terminal fragment of AreA can bind individually to GATA-containing DNA and NmrA and that in the presence of a mixture of GATA-containing DNA and NmrA, the AreA fragment binds preferentially to the GATA-containing DNA in vitro. These observations are consistent with NmrA acting by an indirect route, such as by controlling entry into the nucleus. Deletion of the final nine amino acids of a C-terminal fragment of AreA does not affect NmrA binding. Wild-type NmrA binds NAD(+)(P+) with much greater affinity than NAD(P)H, despite the lack of the consensus GXXGXXG dinucleotide-binding motif. However, introducing the GXXGXXG sequence into the NmrA double mutant N12G/A18G causes an approximately 13-fold increase in the KD for NAD+ and a 2.3-fold increase for NADP+. An H37W mutant in NmrA designed to increase the interaction with the adenine ring of NAD+ has a decrease in KD of approximately 4.5-fold for NAD+ and a marginal 24% increase for NADP+. The crystal structure of the N12G/A18G mutant protein shows changes in main chain position as well as repositioning of H37, which disrupts contacts with the adenine ring of NAD+, changes which are predicted to reduce the binding affinity for this dinucleotide. The substitutions E193Q/D195N or Q202E/F204Y in the C-terminal domain of NmrA reduced the affinity for a C-terminal fragment of AreA, implying that this region of the protein interacts with AreA.

Base Sequence↗

PubMed2004

Crystallographic studies of shikimate binding and induced conformational changes in Mycobacterium tuberculosis shikimate kinase.

The X-ray crystal structure of Mycobacterium tuberculosis shikimate kinase (SK) with bound shikimate and adenosine diphosphate (ADP) has been determined to a resolution of 2.15 A. The binding of shikimate in a shikimate kinase crystal structure has not previously been reported. The substrate binds in a pocket lined with hydrophobic residues and interacts with several highly conserved charged residues including Asp34, Arg58, Glu61 and Arg136 which project into the cavity. Comparisons of our ternary SK-ADP-shikimate complex with an earlier binary SK-ADP complex show that conformational changes occur on shikimate binding with the substrate-binding domain rotating by 10 degrees. Detailed knowledge of shikimate binding is an important step in the design of inhibitors of SK, which have potential as novel anti-tuberculosis agents.

Cloning, Molecular↗

PubMed2004

The phenylmethylthiazolylthiourea nonnucleoside reverse transcriptase (RT) inhibitor MSK-076 selects for a resistance mutation in the active site of human immunodeficiency virus type 2 RT.

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

Amino Acid Sequence↗

PubMed2003

Crystal structure of varicella zoster virus thymidine kinase.

Herpes virus thymidine kinases are responsible for the activation of nucleoside antiviral drugs including (E)-5-(2-bromovinyl)-2'-deoxyuridine. Such viral thymidine kinases (tk), beside having a broader substrate specificity compared with host cell enzymes, also show significant variation in nucleoside phosphorylation among themselves. We have determined the crystal structure of Varicella zoster virus (VZV, human herpes virus 3) thymidine kinase complexed with (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-monophosphate and ADP. Differences in the conformation of a loop region (residues 55-61) and the position of two alpha-helices at the subunit interface of VZV-tk compared with the herpes simplex virus type 1 (human herpes virus 1) enzyme give rise to changes in the positioning of residues such as tyrosine 66 and glutamine 90, which hydrogen bond to the substrate in the active site. Such changes in combination with the substitution in VZV-tk of two phenylalanine residues (in place of a tyrosine and methionine), which sandwich the substrate pyrimidine ring, cause an alteration in the positioning of the base. The interaction of the (E)-5-(2-bromovinyl)-2'-deoxyuridine deoxyribose ring with the protein is altered by substitution of tyrosine 21 and phenylalanine 139 (analagous to herpes simplex virus type 1 histidine 58 and tyrosine 172), which may explain some of the differences in nucleoside sugar selectivity between both enzymes. The altered active site architecture may also account for the differences in the substrate activity of ganciclovir for the two thymidine kinases. These data should be of use in the design of novel antiherpes and antitumor drugs.

Amino Acid Sequence↗

PubMed2003

Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase.

A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.

Cloning, Molecular↗

PubMed2002

Crystal structure of SANOS, a bacterial nitric oxide synthase oxygenase protein from Staphylococcus aureus.

Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 A. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H(4)B) in mammalian NOSs, has NAD(+) bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.

Amino Acid Sequence↗