Document Type
Article
Original Publication Date
2015
Journal/Book/Conference Title
Biochemistry
Volume
54
Issue
38
DOI of Original Publication
10.1021/acs.biochem.5b00610
Date of Submission
December 2015
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.
Rights
Copyright © 2015 American Chemical Society
Is Part Of
VCU Medicinal Chemistry Publications
Figure S1. Structural alignment of AAV serotypes OBD. Figure S2. Sedimentation velocity analysis of OBD and OBDL.
Comments
Originally published at http://dx.doi.org/10.1021/acs.biochem.5b00610