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1.
PLoS Pathog ; 7(5): e1002030, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21589902

RESUMEN

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.


Asunto(s)
Proteínas de la Nucleocápside/química , Multimerización de Proteína , ARN Viral/química , Ribonucleoproteínas/química , Virus de la Fiebre del Valle del Rift/fisiología , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X/métodos , ADN Complementario/genética , Humanos , Microscopía Electrónica , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/aislamiento & purificación , Proteínas de la Nucleocápside/ultraestructura , Dominios y Motivos de Interacción de Proteínas , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/ultraestructura , Virus de la Fiebre del Valle del Rift/química , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/ultraestructura , Alineación de Secuencia , Resonancia por Plasmón de Superficie/métodos , Ensamble de Virus
2.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 65(Pt 10): 1035-8, 2009 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-19851016

RESUMEN

Pseudomonas aeruginosa RocR, an EAL-domain protein which regulates the expression of virulence genes and biofilm formation, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of RocR are reported. The X-ray diffraction data were processed to a resolution of 2.50 A. The crystals belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = 118.8, b = 118.8, c = 495.1 A, alpha = beta = 90, gamma = 120 degrees .


Asunto(s)
Pseudomonas aeruginosa/química , Factores de Transcripción/química , Cristalización , Cristalografía por Rayos X
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