Structure and Dynamics of the P7 Protein from the Bacteriophage ϕ12

Abstract

Cystoviruses are a class of enveloped double-stranded RNA viruses that use a multi-protein polymerase complex (PX) to replicate and transcribe the viral genome. Though the structures of the polymerase and ATPase components of the cystoviral PX are known and their functional behavior understood to a large extent, no atomic-resolution structural information is available for the major capsid protein P1 that defines the overall structure and symmetry of the viral capsid and the essential protein P7. Towards obtaining a complete structural and functional understanding of the cystoviral PX, we have obtained the structure of P7 from the cystovirus ϕ12 at a resolution of 1.8 Å. The N-terminal core region (1–129) of P7 forms a novel homodimeric α/β-fold with structural similarities with BRCT domains implicated in multiple protein-protein interactions in DNA repair proteins. Our results combined with the known role of P7 in stabilizing the nucleation complex during capsid assembly hints towards its participation in key protein-protein interactions within the cystoviral PX. Additionally, we have found through solution NMR studies that the C-terminal tail of P7 (130–169) that is essential for virus viability, though highly disordered, contains a nascent helix. We demonstrate for the very first time, through NMR titrations, that P7 is capable of interacting with RNA. We find that both the N-terminal core and the dynamic C-terminus tail of P7 play a role in RNA recognition leading to a significant reduction of the degree of disorder in the C-terminal tail. Given the requirement of P7 in maintaining genome packaging efficiency and transcriptional fidelity, our data suggest a central biological role for P7/RNA interactions.

Packaging Accessory Protein P7 and Polymerase P2 Have Mutually Occluding Binding Sites inside the Bacteriophage ϕ6 Procapsid

“…The crystal structure of the N-terminal 129 residues of P7 from 12 features a ␤-sheet core surrounded by helices (6). Its elongated shape matches quite well with the molecular envelope assigned to P7 in our difference maps (Fig.…”

“…As the P4 motor threads the RNA strand through the pore, the RNA could interact with P7 inside the shell. Both the N-terminal core and the C-terminal tail have been implicated in RNA binding (6). One possibility is that P7 may provide a catch to prevent retraction of incoming RNA strands during packaging.…”

“…P7 is reported to form dimers in solution (13), and it has been proposed, on the basis of small-angle neutron scattering data, to be positioned at a radius of about 160 Å from the procapsid center (11). The structure of P7 from the related phage 12 has been solved by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, also as a dimer (6), and it has been proposed, based on interpretation of a cryo-electron microscopy (cryo-EM) reconstruction of mature 12 virions, that P7 is situated next to the P4 hexamer, outside the P1 shell (36).…”

Variable Region Identical Immunoglobulins Differing in Isotype Express Different Paratopes

“…There are a variety of extensively used NMR techniques and approaches that can be used to stably label either the Ab or the Ag with isotopes (11,(22)(23)(24) for mapping residue-specific protein-ligand interactions (25,26 …”

Rescue of Maturation Off-Pathway Products in the Assembly of Pseudomonas Phage ϕ6

“…The assembly pathway of these bacterial double-stranded RNA (dsRNA) viruses is particularly intriguing due to the complexity of their genome encapsidation pathway and the multiple enzymatic functions that are associated with assembly intermediates (2)(3)(4). Furthermore, the description of the assembly process down to an atomic level is facilitated by the high-resolution structures of key enzymatic and structural subunits (5)(6)(7)(8)(9)(10).…”