Structural basis for the attachment of a paramyxoviral polymerase to its template

Abstract

The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain. We have investigated the interaction between the binding domain from measles P (amino acids 457-507) and the sequence it recognizes within measles N (amino acids 477-505). By using both solution NMR spectroscopy and x-ray crystallography, we show that N487-503 binds as a helix to the surface created by the second (␣2) and third (␣3) helices of P457-507, in an orientation parallel to the helix ␣3, creating a four-helix bundle. The binding interface is tightly packed and dominated by hydrophobic amino acids. Binding and folding of N487-503 are coupled. However, when not bound to P, N487-503 does not resemble a statistical random coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N487-503 is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase-template interactions in a paramyxovirus and presents an example of a protein-protein interaction that must be only transiently maintained as part of its normal function.M easles virus, a member of the paramyxovirus family, causes an acute infectious disease in humans. The virus is enveloped and possesses a negative-sense, single-stranded RNA genome Ϸ15,900 nt in length. Within the virion, the nucleocapsid protein (N) packages the genomic RNA into a helical protein-RNA complex termed the nucleocapsid. In the cytoplasm of an infected cell, the viral RNA polymerase uses the nucleocapsid as a template for both the transcription of messenger RNAs, encoding the individual viral proteins, as well as the replication and encapsidation of full-length copies of the viral genome. Unencapsidated RNA cannot act as a template for the polymerase. The replication of paramyxoviruses has been comprehensively reviewed (1-3).The paramyxoviral polymerase has two components, the large protein and the phosphoprotein (P) (Fig. 1). The catalytic activities of the polymerase reside within the large protein, whereas P is responsible for, among other activities, binding the polymerase to the nucleocapsid (4, 5). P is a modular protein containing a number of functional domains separated by intrinsically disordered sequences (6). A coiled-coil domain oligomerizes P (7) while the extreme carboxyl terminus of P is involved in nucleocapsid binding. The structure of this region of measles P (amino acids 459-507) has been recently determined by x-ray crystallography and is a compact bundle of three ␣-helices (8). This region...

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

“…analogy with the related MeV (44,74) and also based on ITC studies carried out in the presence of 1 M NaCl, which showed that the N TAIL -P XD interaction does not rely on polar contacts, we reasoned that the burying of apolar residues of N TAIL at the P XD surface could be the driving force in the P XD -induced folding of N TAIL . Indeed, although globular proteins contribute most of their hydrophobic residues to the protein core, IDPs expose their few hydrophobic residues to the surface, thereby allowing interaction with binding partners.…”

“…The amino acid sequence of P XD and of the N TAIL region that was modeled in the complex is shown with the same color code. C, superimposition of the structural models (ribbon representation) of the P XD -N TAIL complexes of HeV (green) and NiV (orange) onto the crystal structure of a MeV chimeric construct (red) encompassing P XD (amino acids 459 -507 of P) and residues 486 -504 of N (PDB code 1T6O) (74). A multiple sequence alignment of Henipavirus and MeV P XD as obtained using ClustalW and ESPript is also shown.…”

“…After having obtained the structural model of both P XD proteins, we modeled the ␣-helical ␣-MoRE of N TAIL in the hydrophobic cleft delimited by helices ␣2 and ␣3 of P XD to yield a pseudo-four-helix arrangement similar to that already observed for the closely related MeV N TAIL -P XD complex (74) (Fig. 8).…”

“…We therefore modeled the more hydrophobic side of the amphipathic ␣-MoRE of N TAIL at the hydrophobic surface delimited by helices ␣2 and ␣3 of P XD (74). We emphasize that the proposed models are only a tentative description of a possible mode of interaction.…”

Plasticity in Structural and Functional Interactions between the Phosphoprotein and Nucleoprotein of Measles Virus

“…a disorder-to-order transition upon binding to a partner) (41)(42)(43)(44). Several studies have confirmed the involvement of this ␣-MoRE in binding to XD, showing that the N TAIL region encompassing residues 486 -502 adopts an ␣-helical conformation when bound to XD (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)45). The ␣-MoRE is partly preconfigured in solution prior to XD binding (21, 24 -26), and binding to XD stabilizes the ␣-helical conformation, giving rise to a compact four-helix bundle in which the N TAIL -XD interface is stabilized by hydrophobic contacts (17,45).…”