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1.
Nat Commun ; 13(1): 5884, 2022 10 06.
Article in English | MEDLINE | ID: mdl-36202813

ABSTRACT

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.


Subject(s)
Cullin Proteins , Molecular Dynamics Simulation , Cullin Proteins/metabolism , Deuterium , Lysine/metabolism , Mass Spectrometry , Proteolysis , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
2.
PLoS One ; 16(10): e0258052, 2021.
Article in English | MEDLINE | ID: mdl-34634061

ABSTRACT

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.


Subject(s)
Bacillus thuringiensis/metabolism , Bacterial Proteins/pharmacology , Insecticides/pharmacology , Pest Control, Biological/methods , Plants, Genetically Modified , Zea mays , Animals , Bacterial Proteins/genetics , Coleoptera/drug effects , Insecticide Resistance/drug effects , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Zea mays/genetics , Zea mays/metabolism
3.
Protein Sci ; 29(4): 824-829, 2020 04.
Article in English | MEDLINE | ID: mdl-31840313

ABSTRACT

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full-length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening-over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate-binding domains, lectins, or from other insecticidal proteins.


Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Protein Folding
4.
Pest Manag Sci ; 76(3): 1031-1038, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31503398

ABSTRACT

BACKGROUND: Protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides act by inhibiting a key enzyme in the heme and chlorophyll biosynthetic pathways in plants. This enzyme, the PPO enzyme, is conserved across plant species. However, some microbes are known to utilize a unique family of PPO enzymes, the HemG family. This enzyme family carries out the same enzymatic step as the plant PPO enzymes, but does not share sequence homology with the plant PPO enzymes. RESULTS: Bioinformatic analysis was used to identify putative HemG PPO enzyme variants from microbial sources. A subset of these variants was cloned and characterized. HemG PPO variants were characterized for functionality and tolerance to PPO-inhibiting herbicides. HemG PPO variants that exhibited insensitivity to PPO-inhibiting herbicides were identified for further characterization. Expression of selected variants in maize, soybean, cotton and canola resulted in plants that displayed tolerance to applications of PPO-inhibiting herbicides. CONCLUSION: Selected microbial-sourced HemG PPO enzyme variants present an opportunity for building new herbicide tolerance biotechnology traits. These traits provide tolerance to PPO-inhibiting herbicides and, therefore, could provide additional tools for farmers to employ in their weed management systems. © 2019 Society of Chemical Industry.


Subject(s)
Biotechnology , Herbicides , Protoporphyrinogen Oxidase , Glycine max , Zea mays
5.
Pest Manag Sci ; 75(8): 2086-2094, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30828945

ABSTRACT

BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.


Subject(s)
Glycine max/enzymology , Herbicide Resistance/genetics , Herbicides/pharmacology , Plants, Genetically Modified/enzymology , Zea mays/enzymology , Genetic Engineering , Indoleacetic Acids/pharmacology , Plants, Genetically Modified/genetics , Propionates/pharmacology , Glycine max/genetics , Zea mays/genetics
6.
Insect Biochem Mol Biol ; 105: 79-88, 2019 02.
Article in English | MEDLINE | ID: mdl-30605769

ABSTRACT

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.


Subject(s)
Bacterial Proteins/metabolism , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Animals , Bacillus thuringiensis Toxins , Insect Control/methods
7.
Nature ; 533(7601): 58-63, 2016 May 05.
Article in English | MEDLINE | ID: mdl-27120167

ABSTRACT

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Directed Molecular Evolution/methods , Endotoxins/genetics , Endotoxins/metabolism , Genetic Variation/genetics , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Insecticide Resistance , Moths/physiology , Pest Control, Biological/methods , Amino Acid Sequence , Animals , Bacillus thuringiensis Toxins , Bacteriophages/genetics , Biotechnology , Cadherins/metabolism , Cell Death , Consensus Sequence , Crops, Agricultural/genetics , Crops, Agricultural/metabolism , Insecticides/metabolism , Molecular Sequence Data , Moths/cytology , Mutagenesis/genetics , Plants, Genetically Modified , Protein Binding/genetics , Protein Stability , Selection, Genetic
8.
Arch Biochem Biophys ; 600: 1-11, 2016 06 15.
Article in English | MEDLINE | ID: mdl-27001423

ABSTRACT

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/ultrastructure , Endotoxins/chemistry , Hemolysin Proteins/chemistry , Hemolysin Proteins/ultrastructure , Heteroptera/chemistry , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/ultrastructure , Saliva/chemistry , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/toxicity , Binding Sites , Endotoxins/toxicity , Hemolysin Proteins/toxicity , Insect Proteins , Pore Forming Cytotoxic Proteins/toxicity , Protein Binding , Protein Conformation , Survival , Trypsin/chemistry
9.
Bone ; 81: 478-486, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26318908

ABSTRACT

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.


Subject(s)
Diphosphonates/chemistry , Geranyltranstransferase/antagonists & inhibitors , Mutation , Nitrogen/chemistry , Bone Density Conservation Agents/therapeutic use , Catalysis , Catalytic Domain , Crystallization , Diphosphonates/therapeutic use , Drug Evaluation, Preclinical , Geranyltranstransferase/chemistry , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Imidazoles/therapeutic use , Inhibitory Concentration 50 , Molecular Conformation , Oligonucleotides/chemistry , Protein Binding , Recombinant Proteins/chemistry , Threonine/chemistry , Tyrosine/chemistry , Zoledronic Acid
10.
Protein Sci ; 23(11): 1491-7, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25139047

ABSTRACT

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.


Subject(s)
Bacterial Proteins/chemistry , Endotoxins/chemistry , Hemolysin Proteins/chemistry , Insecticides/chemistry , Bacillus thuringiensis Toxins , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry
11.
Cell Rep ; 2(3): 518-25, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22999936

ABSTRACT

HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding "P loop motif." How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable "in-out" conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the "out state," which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.


Subject(s)
Drug Resistance, Multiple, Bacterial/physiology , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Protein Serine-Threonine Kinases/chemistry , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Escherichia coli/chemistry , Escherichia coli Proteins/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/metabolism , Serine/chemistry , Serine/metabolism
12.
Arch Biochem Biophys ; 528(1): 90-101, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22750542

ABSTRACT

In this paper we describe the expression, purification, kinetics and biophysical characterization of alanine aminotransferase (AlaAT) from the barley plant (Hordeum vulgare). This dimeric PLP-dependent enzyme is a pivotal element of several key metabolic pathways from nitrogen assimilation to carbon metabolism, and its introduction into transgenic plants results in increased yield. The enzyme exhibits a bi-bi ping-pong reaction mechanism with a K(m) for alanine, 2-oxoglutarate, glutamate and pyruvate of 3.8, 0.3, 0.8 and 0.2 mM, respectively. Barley AlaAT catalyzes the forward (alanine-forming) reaction with a k(cat) of 25.6 s(-1), the reverse (glutamate-forming) reaction with k(cat) of 12.1 s(-1) and an equilibrium constant of ~0.5. The enzyme is also able to utilize aspartate and oxaloacetate with ~10% efficiency as compared to the native substrates, which makes it much more specific than related bacterial/archaeal enzymes (that also have lower K(m) values). We have crystallized barley AlaAT in complex with PLP and l-cycloserine and solved the structure of this complex at 2.7 Å resolution. This is the first example of a plant AlaAT structure, and it reveals a canonical aminotransferase fold similar to structures of the Thermotoga maritima, Pyrococcus furiosus, and human enzymes. This structure bridges our structural understanding of AlaAT mechanism between three kingdoms of life and allows us to shed some light on the specifics of the catalysis performed by these proteins.


Subject(s)
Alanine Transaminase/chemistry , Alanine Transaminase/metabolism , Hordeum/enzymology , Alanine/metabolism , Alanine Transaminase/isolation & purification , Amino Acid Sequence , Aspartic Acid/metabolism , Crystallography, X-Ray , Hordeum/chemistry , Hordeum/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Sequence Alignment , Substrate Specificity
13.
ACS Med Chem Lett ; 3(3): 187-92, 2012 Mar 08.
Article in English | MEDLINE | ID: mdl-24900455

ABSTRACT

Kynurenine aminotransferase (KAT) II has been identified as a potential new target for the treatment of cognitive impairment associated with schizophrenia and other psychiatric disorders. Following a high-throughput screen, cyclic hydroxamic acid PF-04859989 was identified as a potent and selective inhibitor of human and rat KAT II. An X-ray crystal structure and (13)C NMR studies of PF-04859989 bound to KAT II have demonstrated that this compound forms a covalent adduct with the enzyme cofactor, pyridoxal phosphate (PLP), in the active site. In vivo pharmacokinetic and efficacy studies in rat show that PF-04859989 is a brain-penetrant, irreversible inhibitor and is capable of reducing brain kynurenic acid by 50% at a dose of 10 mg/kg (sc). Preliminary structure-activity relationship investigations have been completed and have identified the positions on this scaffold best suited to modification for further optimization of this novel series of KAT II inhibitors.

14.
Proc Natl Acad Sci U S A ; 107(51): 22002-7, 2010 Dec 21.
Article in English | MEDLINE | ID: mdl-21135211

ABSTRACT

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by ß-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed ß-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.


Subject(s)
Anti-Bacterial Agents/chemistry , Models, Molecular , Penicillin-Binding Proteins/chemistry , Pseudomonas aeruginosa/chemistry , Siderophores/chemistry , beta-Lactams/chemistry , Amino Acids, Aromatic , Anti-Bacterial Agents/therapeutic use , Cross Infection/drug therapy , Cross Infection/microbiology , Crystallography, X-Ray , Humans , Protein Structure, Tertiary , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , beta-Lactams/therapeutic use
15.
J Biomol Screen ; 15(8): 1001-7, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20228278

ABSTRACT

Current methods for high-throughput screening (HTS) use a serial process to evaluate compounds as inhibitors toward a single therapeutic target, but as the demand to reduce screening time and cost continues to grow, one solution is the development of multiplex technology. In this communication, the multiplex assay capability of a mass spectrometry (MS)-based readout system is verified using a kinase and esterase reaction simultaneously. Furthermore, the MS-based readout is shown to be compatible with a typical HTS workflow by identifying and validating several new inhibitors for each enzyme from a small library of compounds. These data confirm that it is possible to monitor inhibition of multiple therapeutic targets with one pass through the compound repository, thus demonstrating the potential for MS-based methods to become a method of choice for HTS of isolated enzymes.


Subject(s)
Enzyme Assays/methods , Enzyme Inhibitors/analysis , Enzyme Inhibitors/isolation & purification , High-Throughput Screening Assays/methods , Mass Spectrometry/methods , Acetylcholinesterase/metabolism , Calibration , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Assays/standards , Enzyme Inhibitors/pharmacology , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/metabolism , High-Throughput Screening Assays/standards , Humans , Inhibitory Concentration 50 , Mass Spectrometry/standards , Models, Biological , Small Molecule Libraries/analysis
16.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 8): 875-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19622872

ABSTRACT

Bacterial persistence is the ability of individual cells to randomly enter a period of dormancy during which the cells are protected against antibiotics. In Escherichia coli, persistence is regulated by the activity of a protein kinase HipA and its DNA-binding partner HipB, which is a strong inhibitor of both HipA activity and hip operon transcription. The crystal structure of the HipBA complex was solved by application of the SAD technique to a mercury derivative. In this article, the fortuitous and interesting effect of mercury soaks on the native HipBA crystals is discussed as well as the intriguing tryptophan-binding pocket found on the HipA surface. A HipA-regulation model is also proposed that is consistent with the available structural and biochemical data.


Subject(s)
DNA-Binding Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Protein Kinase Inhibitors/chemistry , Anti-Bacterial Agents/therapeutic use , Binding Sites , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Drug Resistance, Bacterial , Escherichia coli Infections/drug therapy , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genes, Switch/genetics , Humans , Mercury/metabolism , Operon , Protein Conformation , Protein Kinase Inhibitors/metabolism , Structure-Activity Relationship , Tryptophan/metabolism
17.
Protein Sci ; 17(10): 1706-18, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18725455

ABSTRACT

Bacterial acetyl-CoA carboxylase is a multifunctional biotin-dependent enzyme that consists of three separate proteins: biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Acetyl-CoA carboxylase is a potentially attractive target for novel antibiotics because it catalyzes the first committed step in fatty acid biosynthesis. In the first half-reaction, BC catalyzes the ATP-dependent carboxylation of BCCP. In the second half-reaction, the carboxyl group is transferred from carboxybiotinylated BCCP to acetyl-CoA to produce malonyl-CoA. A series of structures of BC from several bacteria crystallized in the presence of various ATP analogs is described that addresses three major questions concerning the catalytic mechanism. The structure of BC bound to AMPPNP and the two catalytically essential magnesium ions resolves inconsistencies between the kinetics of active-site BC mutants and previously reported BC structures. Another structure of AMPPNP bound to BC shows the polyphosphate chain folded back on itself, and not in the correct (i.e., extended) conformation for catalysis. This provides the first structural evidence for the hypothesis of substrate-induced synergism, which posits that ATP binds nonproductively to BC in the absence of biotin. The BC homodimer has been proposed to exhibit half-sites reactivity where the active sites alternate or "flip-flop" their catalytic cycles. A crystal structure of BC showed the ATP analog AMPPCF(2)P bound to one subunit while the other subunit was unliganded. The liganded subunit was in the closed or catalytic conformation while the unliganded subunit was in the open conformation. This provides the first structural evidence for half-sites reactivity in BC.


Subject(s)
Biotin/chemistry , Carbon-Nitrogen Ligases/chemistry , Binding Sites , Carbon-Nitrogen Ligases/antagonists & inhibitors , Catalysis , Crystallization , Crystallography, X-Ray , Escherichia coli/enzymology , Magnesium/chemistry , Nucleotides/chemistry , Pseudomonas aeruginosa/enzymology , Staphylococcus aureus/enzymology
18.
Bioorg Med Chem Lett ; 18(9): 2878-82, 2008 May 01.
Article in English | MEDLINE | ID: mdl-18434151

ABSTRACT

The complex formed from crystallization of human farnesyl pyrophosphate synthase (hFPPS) from a solution of racemic [6,7-dihydro-5H-cyclopenta[c]pyridin-7-yl(hydroxy)methylene]bis(phosphonic acid) (NE-10501, 8), a chiral analog of the anti-osteoporotic drug risedronate, contained the R enantiomer in the enzyme active site. This enantiospecificity was assessed by computer modeling of inhibitor-active site interactions using Autodock 3, which was also evaluated for predictive ability in calculations of the known configurations of risedronate, zoledronate, and minodronate complexed in the active site of hFPPS. In comparison with these structures, the 8 complex exhibited certain differences, including the presence of only one Mg(2+), which could contribute to its 100-fold higher IC(50). An improved synthesis of 8 is described, which decreases the number of steps from 12 to 8 and increases the overall yield by 17-fold.


Subject(s)
Bone Density Conservation Agents/pharmacology , Computer Simulation , Enzyme Inhibitors/pharmacology , Etidronic Acid/analogs & derivatives , Farnesyltranstransferase/antagonists & inhibitors , Organophosphonates/pharmacology , Pyridines/pharmacology , Algorithms , Binding Sites , Bone Density Conservation Agents/chemical synthesis , Carcinoma/drug therapy , Carcinoma/enzymology , Crystallography, X-Ray , Diphosphonates/chemistry , Diphosphonates/pharmacology , Enzyme Inhibitors/chemical synthesis , Etidronic Acid/chemistry , Etidronic Acid/pharmacology , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Inhibitory Concentration 50 , Magnesium/chemistry , Magnesium/metabolism , Models, Chemical , Organophosphonates/chemical synthesis , Pyridines/chemical synthesis , Risedronic Acid , Stereoisomerism , Structure-Activity Relationship , Zoledronic Acid
19.
J Struct Biol ; 162(1): 152-69, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18086534

ABSTRACT

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.


Subject(s)
Bacterial Proteins/chemistry , Phenylalanine-tRNA Ligase/chemistry , Staphylococcus haemolyticus/enzymology , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray/methods , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis , Phenylalanine-tRNA Ligase/antagonists & inhibitors , Phenylalanine-tRNA Ligase/metabolism , Protein Engineering/methods , Protein Structure, Secondary , Protein Structure, Tertiary , Staphylococcus haemolyticus/genetics
20.
Proteins ; 66(3): 538-46, 2007 Feb 15.
Article in English | MEDLINE | ID: mdl-17120228

ABSTRACT

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.


Subject(s)
Bacteria/enzymology , Protease Inhibitors/pharmacology , Aminopeptidases/chemistry , Aminopeptidases/isolation & purification , Bacteria/drug effects , Bacterial Proteins/pharmacology , Crystallization , Crystallography, X-Ray , Kinetics , Magnetic Resonance Spectroscopy , Methionyl Aminopeptidases , Models, Molecular , Protease Inhibitors/chemistry , Protein Conformation , Quantum Theory
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