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Biochemistry. 2015 Sep 29;54(38):5907-19. doi: 10.1021/acs.biochem.5b00610. Epub 2015 Sep 14.

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

Author information

1
Department of Infectious Diseases, King's College London , London SE1 9RT, United Kingdom.
2
UCL Gene Therapy Consortium, UCL Cancer Institute, University College London , London WC1E 6DD, United Kingdom.

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.

PMID:
26314310
PMCID:
PMC4636433
DOI:
10.1021/acs.biochem.5b00610
[Indexed for MEDLINE]
Free PMC Article

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