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1.
J Mol Biol ; 357(2): 665-75, 2006 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-16427083

RESUMEN

Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.


Asunto(s)
Nucleótidos/metabolismo , Conformación Proteica , ARN Polimerasa Dependiente del ARN/química , Ribonucleoproteínas/química , Uridina/metabolismo , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Genoma Viral , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Poliovirus/genética , Poliovirus/metabolismo , ARN Viral , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Alineación de Secuencia , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral
2.
J Mol Biol ; 412(4): 737-50, 2011 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-21839092

RESUMEN

Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the "thumb" domain of one polymerase and the back of the "palm" domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.


Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína/fisiología , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Sustitución de Aminoácidos/fisiología , Modelos Biológicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Poliovirus/enzimología , Poliovirus/genética , Poliovirus/crecimiento & desarrollo , Poliovirus/fisiología , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Mapas de Interacción de Proteínas , Multimerización de Proteína/genética , Estructura Cuaternaria de Proteína/genética , Estructura Secundaria de Proteína/genética , ARN Polimerasa Dependiente del ARN/genética
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