title: Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.
abstract: Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.
authors: Faik N
authors: FN
authors: Musayev
authors: Francisco
authors: F
authors: Zarate-Perez
authors: Department of Infectious Diseases, King's College London , London SE1 9RT, United Kingdom.
authors: Martino
authors: M
authors: Bardelli
authors: Clayton
authors: C
authors: Bishop
authors: Emil F
authors: EF
authors: Saniev
authors: Department of Infectious Diseases, King's College London , London SE1 9RT, United Kingdom.|UCL Gene Therapy Consortium, UCL Cancer Institute, University College London , London WC1E 6DD, United Kingdom.
authors: R Michael
authors: RM
authors: Linden
authors: Department of Infectious Diseases, King's College London , London SE1 9RT, United Kingdom.
authors: Els
authors: E
authors: Henckaerts
authors: Carlos R
authors: CR
authors: Escalante
mesh_terms: D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry*; D000229:Dependovirus / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D010322:Parvoviridae Infections / Q000821:virology; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D014764:Viral Proteins / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2