RESUMO
Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2'-amino-2'-deoxycytidine-5'-triphosphate have been determined to 1.8 A resolution. The alteration of the substrate-binding site that is required to accommodate the 2'-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2'-amino-2'-deoxycytidine-5'-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.
Assuntos
Citidina Trifosfato/análogos & derivados , Inibidores Enzimáticos/metabolismo , Norovirus/química , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citidina Trifosfato/metabolismo , Citidina Trifosfato/farmacologia , Inibidores Enzimáticos/farmacologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Crystal structures of Norwalk virus polymerase bound to an RNA primer-template duplex and either the natural substrate CTP or the inhibitor 5-nitrocytidine triphosphate have been determined to 1.8A resolution. These structures reveal a closed conformation of the polymerase that differs significantly from previously determined open structures of calicivirus and picornavirus polymerases. These closed complexes are trapped immediately prior to the nucleotidyl transfer reaction, with the triphosphate group of the nucleotide bound to two manganese ions at the active site, poised for reaction to the 3'-hydroxyl group of the RNA primer. The positioning of the 5-nitrocytidine triphosphate nitro group between the alpha-phosphate and the 3'-hydroxyl group of the primer suggests a novel, general approach for the design of antiviral compounds mimicking natural nucleosides and nucleotides.
Assuntos
Vírus Norwalk/enzimologia , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Citidina/análogos & derivados , Citidina/química , Primers do DNA/química , Humanos , Picornaviridae/enzimologia , Estrutura Terciária de ProteínaRESUMO
Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.