Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution

9396–9410 Nucleic Acids Research, 2013, Vol. 41, No. 20 doi:10.1093/nar/gkt713 Published online 11 August 2013 Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution Kamel El Omari1, Christoph Meier1, Denis Kainov2,3, Geoff Sutton1, Jonathan M. Grimes1,4, Minna M. Poranen5, Dennis H. Bamford5,6, Roman Tuma7, David I. Stuart1,4 and Erika J. Mancini1,* 1 Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Headington, Oxford OX3 7BN, UK, 2Institute for Molecular Medicine Finland (FIMM), University of Helsinki, 00290 Helsinki, Finland, 3Department of Environmental Research, Siauliai University, Vilniaus gatve_ 88, 76285 Siauliai, Lithuania, 4Diamond Light Source Limited, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK, 5Department of Biosciences, University of Helsinki, Biocenter 2, PO Box 56, 00014 Helsinki, Finland, 6Institute of Biotechnology, University of Helsinki, Biocenter 2, PO Box 56, 00014 Helsinki, Finland and 7Astbury Centre for Structural Molecular Biology and School of Cellular and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Received June 3, 2013; Revised July 18, 2013; Accepted July 19, 2013 ABSTRACT INTRODUCTION Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate singlestranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, r12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from r6, r8 and r13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in r8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in r12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the r12 enzyme. Viruses protect their genome by condensing it into a compartment, the virion. Many complex viruses rely on rapid encapsidation by energy-dependent transport of the nucleic acid into an empty preformed capsid (procapsid). This process requires the presence of portal complexes, which are conduits for nucleic acid molecules, and molecular motors that convert the chemical energy gained from nucleoside triphosphate (NTP) hydrolysis into mechanical movement, resulting in nucleic acid translocation. Some viruses, including herpesvirus and tailed doublestranded DNA (dsDNA) bacteriophages, package their genome using a multi-protein packaging motor (terminase) that transiently assembles at a single vertex (1–4). These complexes are relatively elaborate, consisting of a large dodecameric portal that is an integral part of the capsid and an oligomeric transiently associated terminase, neither of which can work in the absence of the other. The ATPase-nuclease terminase subunit is responsible for recruiting the viral DNA to the procapsid. Compacting relatively stiff dsDNA into a small volume of the procapsid has a high energy cost. Single-molecule experiments have revealed that viral packaging proteins can exert forces as high as 110 pN on dsDNA, making them some of the strongest known biological motors (5). Similarly, dsRNA bacteriophages of the Cystoviridae family (bacteriophages f6 through to f14, and f2954) encapsidate single-stranded RNA (ssRNA) genomic precursors into procapsids (6). However, their packaging *To whom correspondence should be addressed. Tel: +44 1865 287560; Fax: +44 1865 287549; Email: ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2013, Vol. 41, No. 20 9397 machinery is less complex, consisting of a hexamer that is at the same time the physical portal and the active genome translocating motor (7,8). Although this motor shares the same function of translocating the genomic nucleic acid into the procapsid, the challenges differ between ssRNA and dsDNA. ssRNA is significantly more flexible (persistence length lp 1–2 nm) than dsDNA (lp 50 nm) (9), and the packaging densities are less than those found for dsDNA viruses (10); therefore, high forces are probably not required. However, naturally occurring ssRNAs, such as the genomic precursors, exhibit extensive local secondary structure (11,12), and thus the packaging motor has to exhibit helicase activity. The lipid-enveloped bacteriophages of the Cystoviridae family infect Gram-negative bacteria, mainly plant-pathogenic Pseudomonas species (13) and share similarities with the members of the Reoviridae family, including bluetongue virus and rotavirus (14). Their genome of 14 kb consists of three dsRNA segments small (S), medium (M) and large (L), which are sequentially encapsidated as ssRNA precursors into the icosahedrally symmetric procapsid by the packaging NTPase P4 (15–23). P4 NTPases are structural components of the procapsid, built by co-assembly of 120 copies of the major structural protein P1 with 10 copies of the viral RNA-dependent RNA polymerase P2, 10 hexamers of P4 and 12 trimers of the assembly cofactor P7 (24) (Figure 1). In bacteriophage f6, P4 hexamers nucleate procapsid assembly in vitro (7,25), are essential for genome packaging (21) and also have a role in transcription (21,26). Up to 12 P4 hexamers lie on the 5-fold symmetry axes of facets of the procapsid (16,24,27), creating a symmetry mismatch. Although the P4 hexamer constitutes the packaging motor, the specificity for viral RNA is mediated by RNA-binding sites on the P1 shell, which recognize three distinct packaging signals on the genomic precursors (28,29). Previous studies have revealed the structure and mechanism of f12 P4 (30–32). P4 is a protein of 35 kDa, which can assemble into a hexameric ring. NTP-binding sites are located on the external perimeter of the ring at the interfaces between adjacent subunits, whereas the nucleic acid binding sites are found in the central channel (31) (Figure 1). P4 proteins are the only known RNA-specific helicases belonging to helicase Superfamily 4 (SF4) (33). SF4 encompasses mainly DN (...truncated)