The Interdomain Linker of AAV-2 Rep68 Is an Integral Part of Its Oligomerization Domain: Role of a Conserved SF3 Helicase Residue in Oligomerization

PLoS Pathogens

Jorge Mansilla-Soto¹  Demet Kekilli²  Martino Bardelli³  R. Michael Linden³  Kanni Das⁴  John W. Burgner II⁴  Francisco Zarate-Perez⁴  Maria Villamil-Jarauta⁴  Carlos R. Escalante⁵ 

[1] Center for Cell Engineering, Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America;Department of Applied Sciences, University of the West of England, Bristol, United Kingdom;Department of Infectious Diseases, King's College London School of Medicine at Guy's, King's and St. Thomas Hospital, London, United Kingdom;Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America;UCL Gene Therapy Consortium, UCL Cancer Institute, University College London, London, United Kingdom

关键词: Helicases;    Sedimentation;    Protein domains;    Dimers (Chemical physics);    Oligomers;    Nucleotides;    Protein structure;    Protein structure prediction;   

DOI  :  10.1371/journal.ppat.1002764

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

【 授权许可】

CC BY