Binding of Rabies Virus Polymerase Cofactor to Recombinant Circular Nucleoprotein–RNA Complexes

• Published in: Journal of molecular biology 2009-12, Vol.394 (3), p.558-575
• Main Authors: Ribeiro, Euripedes de Almeida, Leyrat, Cédric, Gérard, Francine C.A., Albertini, Aurélie A.V., Falk, Caroline, Ruigrok, Rob W.H., Jamin, Marc
• Format: Article
• Language: English
• Subjects: Amino acids; Binding Sites; Cofactors; Crystal structure; Data processing; Dimerization; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Macromolecular Substances; Models, Molecular; Molds; molecular modeling; Molecular modelling; Neutron Diffraction; Neutron scattering; Nucleocapsid Proteins - chemistry; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - metabolism; Nucleocapsids; Nucleoproteins; Paramyxoviridae - genetics; Paramyxoviridae - metabolism; Phosphoprotein; Phosphoproteins; Protein Structure, Quaternary; Protein Structure, Tertiary; Rabies; Rabies virus; Rabies virus - genetics; Rabies virus - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Replication; Replication complex; Rhabdoviridae - genetics; Rhabdoviridae - metabolism; Rhabdovirus; RNA; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - metabolism; Scattering, Small Angle; Species Specificity; Static Electricity; Surface Plasmon Resonance; Thermodynamics; Titration; Transcription; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; X-Ray Diffraction; X-ray scattering
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2009.09.042

In rabies virus, the attachment of the L polymerase (L) to the viral nucleocapsids (NCs)—a nucleoprotein (N)–RNA complex that serves as template for RNA transcription and replication—is mediated by the polymerase cofactor, the phosphoprotein (P). P forms dimers (P 2) that bind through their C-terminal domains (P CTD) to the C-terminal region of the N. Recombinant circular N m –RNA complexes containing 9 to 12 protomers of N (hereafter, the subscript m denotes the number of N protomers) served here as model systems for studying the binding of P to NC-like N m –RNA complexes. Titration experiments show that there are only two equivalent and independent binding sites for P dimers on the N m –RNA rings and that each P dimer binds through a single P CTD. A dissociation constant in the nanomolar range (160 ± 20 nM) was measured by surface plasmon resonance, indicating a strong interaction between the two partners. Small-angle X-ray scattering (SAXS) data and small-angle neutron scattering data showed that binding of two P CTD had almost no effect on the size and shape of the N m –RNA rings, whereas binding of two P 2 significantly increased the size of the complexes. SAXS data and molecular modeling were used to add flexible loops (N NTD loop, amino acids 105–118; N CTD loop, amino acids 376–397) missing in the recently solved crystal structure of the circular N 11–RNA complex and to build a model for the N 10–RNA complex. Structural models for the N m –RNA–(P CTD) 2 complexes were then built by docking the known P CTD structure onto the completed structures of the circular N 10–RNA and N 11–RNA complexes. A multiple-stage flexible docking procedure was used to generate decoys, and SAXS and biochemical data were used for filtering the models. In the refined model, the P CTD is bound to the C-terminal top of one N protomer (N i), with the C-terminal helix (α 6) of P CTD lying on helix α 14 of N i. By an induced-fit mechanism, the N CTD loop of the same protomer (N i) and that of the adjacent one (N i − 1 ) mold around the P CTD, making extensive protein–protein contacts that could explain the strong affinity of P for its template. The structural model is in agreement with available biochemical data and provides new insights on the mechanism of attachment of the polymerase complex to the NC template.