Structural analysis of the superfamily 3 helicase domain from adeno-associated virus type 2

The non-structural proteins from the SF3 helicase family encode a central or C-terminal NTPase domain to which additional domains are attached to either the N or C-terminus, or both to achieve further specific functions. In the Adeno associated virus type 2 (AAV2) Rep proteins, a central helicase domain is used, along with an N-terminal DNA binding and nicking domain, and a C-terminal zinc finger domain to co-ordinate the activities in the lifecycle of the virus. Events such as replication, integration into host genomes, rescue of integrants, promoter control and viral progeny packaging, are orchestrated by the AAV non-structural Rep proteins; Reps 78, 68, 52 and 40. The smaller of these proteins, Rep40 and 52 are mainly involved in viral DNA packaging.; The Rep40 protein is the core SF3 helicase domain of the larger Rep proteins. In this study, the structure of the Rep40 protein, both with and without nucleotide has been solved. The structures reveal a marked similarity to both the helicase and the AAA+ families of proteins. A hexamer model for Rep40 reveals an arginine finger, thought to be important for inter-subunit communication in oligomeric helicases, and a structural explanation for the role of the conserved Walker B' motif, a conserved feature of the SF3 helicases has also been suggested.; The mode of ADP binding in Rep40 is similar to that observed for other P-loop NTPases. The non-specific interactions of the protein with the adenine ring partially explains the ability of Rep68 and Rep40 to utilize other nucleotides. The nucleotide-binding site is highly polar, and the C4-C5 bond around the ribose moiety is constrained in the atypical gauche-gauche conformation, with the adenine ring of the nucleotide pointing outward from the NTPase core. A nucleotide-binding loop (NB loop) flanks the nucleotide and restricts it into acquiring the observed conformation. In the hexamer model several conserved residues of the Walker B' motif are contributed to the NTPase active site from each neighboring subunit, supporting the conclusion that in the SF3 helicases, the formation of a complete active site for ATP hydrolysis is accomplished by oligomerization.; These structures provide a framework for understanding the structure function relationships in SF3 helicases.