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Nature Structural & Molecular Biology Aug 2016Zika virus has emerged as a pathogen of major health concern. Here, we present a high-resolution (1.62-Å) crystal structure of the RNA helicase from the French...
Zika virus has emerged as a pathogen of major health concern. Here, we present a high-resolution (1.62-Å) crystal structure of the RNA helicase from the French Polynesia strain. The structure is similar to that of the RNA helicase from Dengue virus, with variability in the conformations of loops typically involved in binding ATP and RNA. We identify druggable 'hotspots' that are well suited for in silico and/or fragment-based high-throughput drug discovery.
Topics: Apoenzymes; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; RNA Helicases; Structural Homology, Protein; Viral Proteins; Zika Virus
PubMed: 27399257
DOI: 10.1038/nsmb.3258 -
Signal Transduction and Targeted Therapy Jun 2021
Topics: Apoenzymes; Crystallography, X-Ray; Humans; Ligands; Protein Conformation; Receptors, Serotonin; Serotonin; Serotonin Receptor Agonists
PubMed: 34145221
DOI: 10.1038/s41392-021-00668-3 -
Biophysical Journal Oct 2016Ca-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca-binding event is a prerequisite for membrane association....
Ca-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.
Topics: Allosteric Regulation; Apoenzymes; C2 Domains; Calcium; Hydrogen Bonding; Metals; Models, Molecular; Protein Binding; Protein Kinase C-alpha
PubMed: 27760353
DOI: 10.1016/j.bpj.2016.09.008 -
Biochemistry Dec 2013The crystal structure of formiminoglutamase from Trypanosoma cruzi (TcFIGase) is reported at 1.85 Å resolution. Although the structure of this enzyme was previously...
The crystal structure of formiminoglutamase from Trypanosoma cruzi (TcFIGase) is reported at 1.85 Å resolution. Although the structure of this enzyme was previously determined by the Structural Genomics of Pathogenic Protozoa Consortium (PDB accession code 2A0M), this structure was determined at low pH and lacked bound metal ions; accordingly, the protein was simply annotated as "arginase superfamily protein" with undetermined function. We show that reconstitution of this protein with Mn²⁺ confers maximal catalytic activity in the hydrolysis of formiminoglutamate to yield glutamate and formamide, thereby demonstrating that this protein is a metal-dependent formiminoglutamase. Equilibration of TcFIGase crystals with MnCl₂ at higher pH yields a binuclear manganese cluster similar to that observed in arginase, except that the histidine ligand to the Mn²⁺(A) ion of arginase is an asparagine ligand (N114) to the Mn²⁺(A) ion of TcFIGase. The crystal structure of N114H TcFIGase reveals a binuclear manganese cluster essentially identical to that of arginase, but the mutant exhibits a modest 35% loss of catalytic efficiency (k(cat)/K(M)). Interestingly, when TcFIGase is prepared and crystallized in the absence of reducing agents at low pH, a disulfide linkage forms between C35 and C242 in the active site. When reconstituted with Mn²⁺ at higher pH, this oxidized enzyme exhibits a modest 33% loss of catalytic efficiency. Structure determinations of the metal-free and metal-bound forms of oxidized TcFIGase reveal that although disulfide formation constricts the main entrance to the active site, other structural changes open alternative channels to the active site that may help sustain catalytic activity.
Topics: Amino Acid Sequence; Amino Acid Substitution; Apoenzymes; Arginase; Asparagine; Binding Sites; Conserved Sequence; Cysteine; Formiminoglutamic Acid; Hydrolases; Manganese; Metalloproteins; Models, Molecular; Molecular Sequence Data; Mutant Proteins; Protein Conformation; Protozoan Proteins; Recombinant Proteins; Sequence Alignment; Trypanosoma cruzi
PubMed: 24261485
DOI: 10.1021/bi401352h -
The Journal of Biological Chemistry Aug 2017Hydrogen sulfide (HS) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which...
Hydrogen sulfide (HS) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts HS to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.
Topics: Amino Acid Substitution; Apoenzymes; Bacterial Proteins; Biocatalysis; Burkholderiaceae; Catalytic Domain; Computational Biology; Crystallography, X-Ray; Cysteine; Disulfides; Enzyme Stability; Glutathione; Hydrogen Sulfide; Models, Molecular; Mutant Chimeric Proteins; Mutation; Peptide Fragments; Protein Conformation; Quinone Reductases; Recombinant Proteins; Thiosulfate Sulfurtransferase; Thiosulfates
PubMed: 28684420
DOI: 10.1074/jbc.M117.790170 -
European Journal of Biochemistry Jan 2004Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of... (Comparative Study)
Comparative Study
Mutants of a cobalt-containing nitrile hydratase (NHase, EC 4.2.1.84) from Pseudonocardia thermophila JCM 3095 involved in substrate binding, catalysis and formation of the active center were constructed, and their characteristics and crystal structures were investigated. As expected from the structure of the substrate binding pocket, the wild-type enzyme showed significantly lower K(m) and K(i) values for aromatic substrates and inhibitors, respectively, than aliphatic ones. In the crystal structure of a complex with an inhibitor (n-butyric acid) the hydroxyl group of betaTyr68 formed hydrogen bonds with both n-butyric acid and alphaSer112, which is located in the active center. The betaY68F mutant showed an elevated K(m) value and a significantly decreased k(cat) value. The apoenzyme, which contains no detectable cobalt atom, was prepared from Escherichia coli cells grown in medium without cobalt ions. It showed no detectable activity. A disulfide bond between alphaCys108 and alphaCys113 was formed in the apoenzyme structure. In the highly conserved sequence motif in the cysteine cluster region, two positions are exclusively conserved in cobalt-containing or iron-containing nitrile hydratases. Two mutants (alphaT109S and alphaY114T) were constructed, each residue being replaced with an iron-containing one. The alphaT109S mutant showed similar characteristics to the wild-type enzyme. However, the alphaY114T mutant showed a very low cobalt content and catalytic activity compared with the wild-type enzyme, and oxidative modifications of alphaCys111 and alphaCys113 residues were not observed. The alphaTyr114 residue may be involved in the interaction with the nitrile hydratase activator protein of P. thermophila.
Topics: Actinomycetales; Amino Acid Sequence; Apoenzymes; Binding Sites; Butyric Acid; Catalysis; Cobalt; Conserved Sequence; Crystallization; Cysteine; Disulfides; Escherichia coli; Hydro-Lyases; Hydrogen Bonding; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Binding; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 14717710
DOI: 10.1046/j.1432-1033.2003.03943.x -
Nature Nov 2015Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In...
Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.
Topics: Apoenzymes; Binding Sites; Crystallography, X-Ray; Endonucleases; Enzyme Activation; Gammainfluenzavirus; Models, Molecular; Peptide Chain Initiation, Translational; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Protein Subunits; RNA Caps; RNA, Viral; RNA-Dependent RNA Polymerase; Ribonucleoproteins
PubMed: 26503046
DOI: 10.1038/nature15525 -
European Journal of Biochemistry 1980The reconstructive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin...
The reconstructive ability of the isolated prosthetic group of methanol dehydrogenase with the apoenzyme of glucose dehydrogenase and the results of electron spin resonance measurements suggest that the prosthetic group has not been modified during the isolation. This result, and the properties of the directly isolated prosthetic group and derivatives, confirm the suggestion that its structure is 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione. From the activity shown by derivatives of the prosthetic group and of structural analogues in the apoenzyme test it is concluded that the o-quinone structure is essential for activity. Hence the trivial name pyrrolo-quinoline quinone would be appropriate. The testing of the analogues also shows that the pyrrolo ring and the 9-carboxylic acid group are not essential for activity as they can be replaced by a pyridinol ring and a 9-hydroxy group respectively. The determination of the molar absorption coefficient of the prosthetic group (18 400 M-1 cm-1 at 249 nm) enables its quantitative anaysis. Thus it could be established that methanol dehydrogenase contains one prosthetic group per enzyme molecule. The consequences of this result in relation to already known properties of this 'quinoprotein' dehydrogenase are discussed.
Topics: Alcohol Oxidoreductases; Apoenzymes; Borohydrides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Methylation; Oxidation-Reduction; PQQ Cofactor; Periodic Acid; Pyrroles; Quinolines
PubMed: 6250827
DOI: 10.1111/j.1432-1033.1980.tb04711.x -
The Journal of Biological Chemistry Oct 2011Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and...
Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.
Topics: Acid Anhydride Hydrolases; Adenosine Triphosphatases; Apoenzymes; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Protein Folding; Protein Structure, Secondary; Streptococcus pneumoniae
PubMed: 21865160
DOI: 10.1074/jbc.M111.228585 -
Biochemistry Aug 2016Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the...
Xylanases catalyze the hydrolysis of xylan, an abundant carbon and energy source with important commercial ramifications. Despite tremendous efforts devoted to the catalytic improvement of xylanases, success remains limited because of our relatively poor understanding of their molecular properties. Previous reports suggested the potential role of atomic-scale residue dynamics in modulating the catalytic activity of GH11 xylanases; however, dynamics in these studies was probed on time scales orders of magnitude faster than the catalytic time frame. Here, we used nuclear magnetic resonance titration and relaxation dispersion experiments ((15)N-CPMG) in combination with X-ray crystallography and computational simulations to probe conformational motions occurring on the catalytically relevant millisecond time frame in xylanase B2 (XlnB2) and its catalytically impaired mutant E87A from Streptomyces lividans 66. Our results show distinct dynamical properties for the apo and ligand-bound states of the enzymes. The apo form of XlnB2 experiences conformational exchange for residues in the fingers and palm regions of the catalytic cleft, while the catalytically impaired E87A variant displays millisecond dynamics only in the fingers, demonstrating the long-range effect of the mutation on flexibility. Ligand binding induces enhanced conformational exchange of residues interacting with the ligand in the fingers and thumb loop regions, emphasizing the potential role of residue motions in the fingers and thumb loop regions for recognition, positioning, processivity, and/or stabilization of ligands in XlnB2. To the best of our knowledge, this work represents the first experimental characterization of millisecond dynamics in a GH11 xylanase family member. These results offer new insights into the potential role of conformational exchange in GH11 enzymes, providing essential dynamic information to help improve protein engineering and design applications.
Topics: Amino Acid Substitution; Apoenzymes; Bacterial Proteins; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Endo-1,4-beta Xylanases; Genes, Bacterial; Ligands; Models, Molecular; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Recombinant Proteins; Streptomyces lividans
PubMed: 27387012
DOI: 10.1021/acs.biochem.6b00130