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1.  Crystal Structures of Type I Dehydroquinate Dehydratase in Complex with Quinate and Shikimate Suggest a Novel Mechanism of Schiff Base Formation 
Biochemistry  2014;53(5):872-880.
A component of the shikimate biosynthetic pathway, dehydroquinate dehydratase (DHQD) catalyzes the dehydration of 3-dehydroquniate (DHQ) to 3-dehydroshikimate. In the type I DHQD reaction mechanism a lysine forms a Schiff base intermediate with DHQ. The Schiff base acts as an electron sink to facilitate the catalytic dehydration. To address the mechanism of Schiff base formation, we determined structures of the Salmonella enterica wild-type DHQD in complex with the substrate analogue quinate and the product analogue shikimate. In addition, we determined the structure of the K170M mutant (Lys170 being the Schiff base forming residue) in complex with quinate. Combined with nuclear magnetic resonance and isothermal titration calorimetry data that revealed altered binding of the analogue to the K170M mutant, these structures suggest a model of Schiff base formation characterized by the dynamic interplay of opposing forces acting on either side of the substrate. On the side distant from the substrate 3-carbonyl group, closure of the enzyme’s β8−α8 loop is proposed to guide DHQ into the proximity of the Schiff base-forming Lys170. On the 3-carbonyl side of the substrate, Lys170 sterically alters the position of DHQ’s reactive ketone, aligning it at an angle conducive for nucleophilic attack. This study of a type I DHQD reveals the interplay between the enzyme and substrate required for the correct orientation of a functional group constrained within a cyclic substrate.
doi:10.1021/bi4015506
PMCID: PMC3985847  PMID: 24437575
2.  Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus 
Applied and Environmental Microbiology  2014;80(13):3992-4002.
Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD+ binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.
doi:10.1128/AEM.00215-14
PMCID: PMC4054205  PMID: 24747910
3.  Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition 
Structure(London, England:1993)  2014;22(7):949-960.
Summary
Peptidoglycan surrounds the bacterial cytoplasmic membrane to protect the cell against osmolysis. The biosynthesis of peptidoglycan, made of glycan strands crosslinked by short peptides, is the target of antibiotics like β-lactams and glycopeptides. Nascent peptidoglycan contains pentapeptides that are trimmed by carboxypeptidases to tetra- and tripeptides. The well-characterized DD-carboxypeptidases hydrolyze the terminal D-alanine from the stem pentapeptide to produce a tetrapeptide. However, few LD-carboxypeptidases that produce tripeptides have been identified, and nothing is known about substrate specificity in these enzymes. We report biochemical properties and crystal structures of the LD-carboxypeptidases LdcB from Streptococcus pneumoniae, Bacillus anthracis, and Bacillus subtilis. The enzymes are active against bacterial cell wall tetrapeptides and adopt a zinc-carboxypeptidase fold characteristic of the LAS superfamily. We have also solved the structure of S. pneumoniae LdcB with a product mimic, elucidating the residues essential for peptidoglycan recognition and the conformational changes that occur on ligand binding.
Highlights
•A peptidoglycan, peptide stem-trimming carboxypeptidase, LdcB, has been characterized•The crystal structure of LdcB has been solved with a peptidoglycan mimic bound•The LdcB structure undergoes significant conformational change on binding ligand•The exquisite substrate specificity of LdcB has also been demonstrated in vitro
Peptidoglyan is an essential layer surrounding the bacterial cytoplasmic membrane that is matured and trimmed by carboxypeptidases. Hoyland et al. describe the structure of one such carboxypeptidase in the presence of a product mimic, explaining the molecular specificity of the enzyme family.
doi:10.1016/j.str.2014.04.015
PMCID: PMC4087270  PMID: 24909784
4.  Targeting DXP synthase in human pathogens: enzyme inhibition and antimicrobial activity of butylacetylphosphonate 
The Journal of antibiotics  2013;67(1):77-83.
The unique methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most bacterial pathogens. The first enzyme in this pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes a distinct thiamin diphosphate (ThDP)-dependent reaction to form DXP from D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate and represents a potential anti-infective drug target. We have previously demonstrated that the unnatural bisubstrate analog, butylacetylphosphonate (BAP), exhibits selective inhibition of Escherichia coli DXP synthase over mammalian ThDP-dependent enzymes. Here, we report the selective inhibition by BAP against recombinant DXP synthase homologs from Mycobacterium tuberculosis, Yersinia pestis, and Salmonella enterica. We also demonstrate antimicrobial activity of BAP against both Gram-negative and Gram-positive strains (including E. coli, S. enterica, Bacillus anthracis), and several clinically isolated pathogens. Our results suggest a mechanism of action involving inhibition of DXP synthase and show that BAP acts synergistically with established antimicrobial agents, highlighting a potential strategy to combat emerging resistance in bacterial pathogens.
doi:10.1038/ja.2013.105
PMCID: PMC3946878  PMID: 24169798
DXP synthase; isoprenoid biosynthesis; selective inhibitor; thiamin diphosphate
5.  Virtual High-Throughput Ligand Screening 
In Structural Genomics projects, virtual high-throughput ligand screening can be utilized to provide important functional details for newly determined protein structures. Using a variety of publicly available software tools, it is possible to computationally model, predict, and evaluate how different ligands interact with a given protein. At the Center for Structural Genomics of Infectious Diseases (CSGID) a series of protein analysis, docking and molecular dynamics software is scripted into a single hierarchical pipeline allowing for an exhaustive investigation of protein-ligand interactions. The ability to conduct accurate computational predictions of protein-ligand binding is a vital component in improving both the efficiency and economics of drug discovery. Computational simulations can minimize experimental efforts, the slowest and most cost prohibitive aspect of identifying new therapeutics.
doi:10.1007/978-1-4939-0354-2_19
PMCID: PMC4073479  PMID: 24590723
Protein; Ligand; High-throughput screening; Docking; Molecular modeling
6.  Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation 
PLoS ONE  2014;9(4):e94816.
The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.
doi:10.1371/journal.pone.0094816
PMCID: PMC3995681  PMID: 24756028
7.  Acetylation of the Response Regulator RcsB Controls Transcription from a Small RNA Promoter 
Journal of Bacteriology  2013;195(18):4174-4186.
Nε-lysine acetylation was recently discovered on many bacterial proteins that function in diverse cellular processes. Thus, many questions remain unanswered. For example, what mechanisms regulate lysine acetylation? Does acetylation affect physiology? To help answer these questions, we studied the Escherichia coli response regulator and transcription factor RcsB, which is reported to be acetylated in vitro. To characterize RcsB acetylation, we monitored transcription from the rprA promoter, which requires RcsB. The conventional view is that RcsB is activated by phosphorylation through either the Rcs phosphorelay or acetyl phosphate. We affirmed that rprA transcription requires phosphorylated RcsB and showed that acetyl-phosphate (AcP) is a phosphoryl group donor to RcsB. However, a mutant that accumulates AcP (ackA) exhibited a reduction in rprA transcription instead of the predicted increase. rprA transcription also diminished in the cobB mutant, which lacks the only known E. coli protein deacetylase. This suggests the existence of an inhibitory mechanism that involves lysine acetylation, a supposition supported by the observation that RcsB isolated from the ackA or cobB mutant was hyperacetylated. Finally, we used a genetic approach to identify an AckA- and CobB-sensitive lysine (Lys-154) that controls RcsB activity. We propose that acetylation inhibits RcsB activity and that some of this inhibition acts through the acetylation of Lys-154.
doi:10.1128/JB.00383-13
PMCID: PMC3754749  PMID: 23852870
8.  Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila 
Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.
Electronic supplementary material
The online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s10969-014-9176-z
PMCID: PMC3955178  PMID: 24562475
Trimethylamine; NTF2-like superfamily; Caci_0382; X-ray crystal structure; Structural genomics
9.  Discovery of Selective Inhibitors of the Clostridium difficile Dehydroquinate Dehydratase 
PLoS ONE  2014;9(2):e89356.
A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3rd of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.
doi:10.1371/journal.pone.0089356
PMCID: PMC3931744  PMID: 24586713
11.  Crystal structure of a type II dehydroquinate dehydratase-like protein from Bifidobacterium longum 
Journal of structural and functional genomics  2013;14(1):10.1007/s10969-013-9149-7.
Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. Here we identify a Bifidobacterium longum protein with high sequence homology to type II DHQDs but no detectable DHQD activity under standard assay conditions. A crystal structure reveals that the B. longum protein adopts a DHQD-like tertiary structure but a distinct quaternary state. Apparently forming a dimer, the B. longum protein lacks the active site aspartic acid contributed from a neighboring protomer in the type II DHQD dodecamer. Relating to the absence of protein–protein interactions established in the type II DHQD dodecameric assembly, substantial conformational changes distinguish the would-be active site of the B. longum protein. As B. longum possess no other genes with homology to known DHQDs, these findings imply a unique DHQD activity within B. longum.
doi:10.1007/s10969-013-9149-7
PMCID: PMC3878977  PMID: 23539270
Post-translational activation; Quaternary structure; Shikimate pathway; X-ray crystal structure; Structural genomics
12.  Correction: A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4 
PLoS ONE  2013;8(12):10.1371/annotation/5edeb1de-b76c-4fd9-a3f6-0f1cf45e3905.
doi:10.1371/annotation/5edeb1de-b76c-4fd9-a3f6-0f1cf45e3905
PMCID: PMC3875687
13.  A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4 
PLoS ONE  2013;8(12):e81504.
Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors.
doi:10.1371/journal.pone.0081504
PMCID: PMC3855329  PMID: 24339940
14.  The crystal structures of the α-subunit of the α2β2 tetrameric Glycyl-tRNA synthetase 
Aminoacyl-tRNA synthetases (AARSs) are ligases (EC.6.1.1.-) that catalyze the acylation of amino acids to their cognate tRNAs in the process of translating genetic information from mRNA to protein. Their amino acid and tRNA specificity are crucial for correctly translating the genetic code. Glycine is the smallest amino acid and the glycyl-tRNA synthetase (GlyRS) belongs to Class II AARSs. The enzyme is unusual because it can assume different quaternary structures. In eukaryotes, archaebac-teria and some bacteria, it forms an α2 homodimer. In some bacteria, GlyRS is an α2β2 heterotetramer and shows a distant similarity to α2 GlyRSs. The human pathogen eubacterium Campylobacter jejuni GlyRS (CjGlyRS) is an α2β2 heterotetramer and is similar to Escherichia coli GlyRS; both are members of Class IIc AARSs. The two-step aminoacylation reaction of tetrameric GlyRSs requires the involvement of both α- and β-subunits. At present, the structure of the GlyRS α2β2 class and the details of the enzymatic mechanism of this enzyme remain unknown. Here we report the crystal structures of the catalytic α-subunit of CjGlyRS and its complexes with ATP, and ATP and glycine. These structures provide detailed information on substrate binding and show evidence for a proposed mechanism for amino acid activation and the formation of the glycyl-adenylate intermediate for Class II AARSs.
doi:10.1007/s10969-012-9142-6
PMCID: PMC3691008  PMID: 23054484
Gly-tRNA synthetase; Catalytic subunit; ATP binding; Glycine binding
15.  Bacillus anthracis Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes 
Biochemistry  2012;51(31):10.1021/bi300511w.
Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed “apo”) form and in complex with its substrate, inosine 5′-monophosphate (IMP), and product, xanthosine 5′-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.
doi:10.1021/bi300511w
PMCID: PMC3836674  PMID: 22788966
16.  LigSearch: a knowledge-based web server to identify likely ligands for a protein target 
LigSearch is a web server for identifying ligands likely to bind to a given protein. It can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.
Identifying which ligands might bind to a protein before crystallization trials could provide a significant saving in time and resources. LigSearch, a web server aimed at predicting ligands that might bind to and stabilize a given protein, has been developed. Using a protein sequence and/or structure, the system searches against a variety of databases, combining available knowledge, and provides a clustered and ranked output of possible ligands. LigSearch can be accessed at http://www.ebi.ac.uk/thornton-srv/databases/LigSearch.
doi:10.1107/S0907444913022294
PMCID: PMC3852652  PMID: 24311580
LigSearch; ligand prediction
17.  Characterization of Fibrinogen Binding by Glycoproteins Srr1 and Srr2 of Streptococcus agalactiae* 
The Journal of Biological Chemistry  2013;288(50):35982-35996.
Background: The serine-rich repeat glycoproteins Srr1 and Srr2 are surface adhesins of Streptococcus agalactiae important for pathogenicity.
Results: Both Srrs bind tandem repeats of the fibrinogen Aα chain, but Srr2 has greater affinity explained by structure-function analysis of the Srrs.
Conclusion: A dock, lock, and latch mechanism describes the Srr-fibrinogen interaction.
Significance: The higher affinity of Srr2 may contribute to the hypervirulence of Srr2-expressing strains.
The serine-rich repeat glycoproteins of Gram-positive bacteria comprise a large family of cell wall proteins. Streptococcus agalactiae (group B streptococcus, GBS) expresses either Srr1 or Srr2 on its surface, depending on the strain. Srr1 has recently been shown to bind fibrinogen, and this interaction contributes to the pathogenesis of GBS meningitis. Although strains expressing Srr2 appear to be hypervirulent, no ligand for this adhesin has been described. We now demonstrate that Srr2 also binds human fibrinogen and that this interaction promotes GBS attachment to endothelial cells. Recombinant Srr1 and Srr2 bound fibrinogen in vitro, with affinities of KD = 2.1 × 10−5 and 3.7 × 10−6 m, respectively, as measured by surface plasmon resonance spectroscopy. The binding site for Srr1 and Srr2 was localized to tandem repeats 6–8 of the fibrinogen Aα chain. The structures of both the Srr1 and Srr2 binding regions were determined and, in combination with mutagenesis studies, suggest that both Srr1 and Srr2 interact with a segment of these repeats via a “dock, lock, and latch” mechanism. Moreover, properties of the latch region may account for the increased affinity between Srr2 and fibrinogen. Together, these studies identify how greater affinity of Srr2 for fibrinogen may contribute to the increased virulence associated with Srr2-expressing strains.
doi:10.1074/jbc.M113.513358
PMCID: PMC3861647  PMID: 24165132
Bacterial Adhesion; Bacterial Pathogenesis; Fibrinogen; Protein Crystallization; Streptococcus; Streptococcus agalactiae; Prot
18.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis 
Journal of molecular biology  2012;420(0):366-383.
Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.
doi:10.1016/j.jmb.2012.04.011
PMCID: PMC3690760  PMID: 22516613
MccF; serine peptidase; nucleophilic elbow; catalytic triad (Ser-His-Glu); substrate binding loop
19.  Development of Novel In Vivo Chemical Probes to Address CNS Protein Kinase Involvement in Synaptic Dysfunction 
PLoS ONE  2013;8(6):e66226.
Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.
doi:10.1371/journal.pone.0066226
PMCID: PMC3694096  PMID: 23840427
20.  Structure of the Type III Secretion Effector Protein ExoU in Complex with Its Chaperone SpcU 
PLoS ONE  2012;7(11):e49388.
Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.
doi:10.1371/journal.pone.0049388
PMCID: PMC3498133  PMID: 23166655
21.  Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria 
The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS.
Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-­ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.
doi:10.1107/S0907444912029101
PMCID: PMC3447402  PMID: 22993090
acyl-carrier-protein synthase; acyl carrier protein; type II fatty-acid synthesis; inhibition; 3′,5′-adenosine diphosphate; coenzyme A
22.  A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding† 
Biochemistry  2011;50(12):2357-2363.
Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD which address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of loop involvement in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, loop closure necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria makes the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.
doi:10.1021/bi102020s
PMCID: PMC3062685  PMID: 21291284
23.  Structural insight into the mechanism of cyclic di-GMP hydrolysis by EAL domain phosphodiesterases 
Journal of molecular biology  2010;402(3):524-538.
Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.
doi:10.1016/j.jmb.2010.07.050
PMCID: PMC2945410  PMID: 20691189
EAL domain; cyclic di-GMP; phosphodiesterase; X-ray crystallography; Thiobacillus denitrificans
24.  Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex* 
The Journal of Biological Chemistry  2011;286(38):33158-33166.
The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn2+. In contrast, with Mg2+ hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.
doi:10.1074/jbc.M111.250456
PMCID: PMC3190883  PMID: 21757692
Allosteric Regulation; Crystal Structure; Enzyme Catalysis; Enzyme Structure; Metalloenzymes; Nucleoside Nucleotide Metabolism; Phosphodiesterases; Deoxynucleotide Triphosphate Triphosphohydrolase
25.  Role of the Membrane Localization Domain of the Pseudomonas aeruginosa Effector Protein ExoU in Cytotoxicity▿ †  
Infection and Immunity  2010;78(8):3346-3357.
ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A2 (PLA2) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA2 activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence.
doi:10.1128/IAI.00223-10
PMCID: PMC2916264  PMID: 20479080

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