Structural basis for the coevolution of a viral RNA–protein complex

Abstract

The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved β-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.The inherent sequence diversity generated by RNA replication, which allows RNA viruses to evolve, must be tempered by the need for conservation of functional elements within the viral genome. The coat proteins of single-stranded (ss) RNA bacteriophages have both structural and regulatory roles in the viral life cycle, as they assemble into the mature capsid and regulate translation of the phage replicase through binding and sequestration of an RNA hairpin that contains the initiation site 1 . The coat protein and translational operator form a pair that must coevolve, so that mutation in one must be accompanied by compensatory mutations in the other. The Pseudomonas aeruginosa bacteriophage PP7 is a model system for exploring coevolution because of the extensive sequence divergence of both the PP7 coat protein and translational operator from other ssRNA bacteriophages 2 . The PP7 and MS2 coat proteins share only 15% sequence identity, and their cognate RNA hairpins differ in the position of the bulged adenosine as well as in the size and nucleotide composition of the loop (Fig. 1a). Biochemical experiments have shown that both coat proteins bind their own RNA hairpins with high affinity (K d ~ 1 nM) and are able to discriminately bind in favor of their own RNA by ~1,000-fold 3,4 . In contrast to the PP7 coat protein, the Qβ coat protein also shares low sequence identity (21%) with the MS2 coat protein, yet it uses an RNAbinding mode similar to that of the MS2 coat protein [5][6][7] Fig. 1 and Supplementary Methods online). PP7ΔFG was generated by removing residues 67-75 and, notably, this truncation binds its translational operator with affinity similar to that of the fulllength protein ( Supplementary Fig. 2 online). The PP7ΔFG monomer adopts the topology characteristic of ssRNA bacteriophage coat proteins, with an N-terminal β hairpin, a fivestranded antiparallel β sheet and two C-terminal α helices ( Supplementary Fig. 3a online) [8][9][10][11] . Antiparallel association of protomers in the dimer positions the last β strands adjacent to one another, resulting in a ten-stranded β sheet that comprises the RNA-binding surface. The interwoven packing of the α helices between each other and the N-terminal β hairpins further stabilizes the dimer, combining with the β sheet interface to bury ~3,000 Å 2 of solvent-accessible surface area. The PP7ΔFG dimer has almost identical free and bound structures (r.m.s. deviation < 0.9 Å for all Cα atoms) and is similar to that of the MS2 coat protein (r.m.s. deviation of < 2.3 Å for all Cα atoms...

Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes

“…Others have used it to study the association of bacteriophage PP7 coat protein and vertebrate ZBP variants to their cognate RNA sequences (Chao et al 2008(Chao et al , 2010. These results show that the method is applicable to a wide variety of protein-RNA complexes.…”

Imaging mRNA and protein interactions within neurons

“…A vector transformation map (affine transformation matrix) was used to then correct the images for chromatic aberration. with high affinity (14,21,22). To confirm the intracellular association between MCP-GFP and single-β-actin mRNA molecules within the cell, we performed single-molecule FISH in combination with immunofluorescence (smFISH-IF) in neurons ( Fig.…”

“…The distance for MCP-GFP to β-actin mRNA is estimated at 7 nm. The drawing of the molecules was generated in PyMol software with the help of published structure data (22,44).…”

Computational Tools and Databases for the Study and Characterization of Protein Interactions

“…Since the seminal work of Altschuh identifying correlated amino acid changes in Tobacco mosaic virus (Altschuh et al 1987), studies recognizing co-evolution as an indicative, albeit subtle, signal of PPIs have being reported in the literature (Travers and Fares 2007;Chao et al 2008;Presser et al 2008). Co-evolutionary information may be divided into three groups: the simultaneous loss or gain of orthologous genes , correlated changes affecting both interacting partners at whole sequence level (explored by mirrortree-based approaches) (Goh and Cohen 2002;Hakes et al 2007;Juan et al 2008) or single amino acids changes (Mintseris and Weng 2005;Madaoui and Guerois 2008).…”