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

Comments

Originally published at http://dx.doi.org/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

NIHMS727457-supplement-supplemental_data.pdf (6158 kB)
Figure S1. Structural alignment of AAV serotypes OBD. Figure S2. Sedimentation velocity analysis of OBD and OBDL.

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