Structural studies of AAV2 Rep68 reveal a partially structured linker and compact domain conformation

TY - JOUR

T1 - Structural studies of AAV2 Rep68 reveal a partially structured linker and compact domain conformation

AU - Musayev, Faik N

AU - Zarate-Perez, Francisco

AU - Bardelli, Martino Raul

AU - Bishop, Clayton M

AU - Saniev, Emil F

AU - Linden, R Michael

AU - Henckaerts, Els

AU - Escalante, Carlos R

PY - 2015/8/28

Y1 - 2015/8/28

N2 - Adeno-associated virus (AAV) non-structural 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 paper 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 acquiring a conformation that positions key residues in all domains on one face of the protein and poised to interact with DNA.

AB - Adeno-associated virus (AAV) non-structural 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 paper 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 acquiring a conformation that positions key residues in all domains on one face of the protein and poised to interact with DNA.

U2 - 10.1021/acs.biochem.5b00610

DO - 10.1021/acs.biochem.5b00610

M3 - Article

C2 - 26314310

SN - 0006-2960

JO - Biochemistry

JF - Biochemistry

ER -