The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: Role of a conserved SF3 helicase residue in oligomerization Journal Article

Authors: Zarate-Perez, F.; Bardelli, M.; Burgner, J. W. 2nd; Villamil-Jarauta, M.; Das, K.; Kekilli, D.; Mansilla-Soto, J.; Linden, R. M.; Escalante, C. R.
Article Title: The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: Role of a conserved SF3 helicase residue in oligomerization
Abstract: 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. © 2012 Zarate-Perez et al.
Keywords: unclassified drug; dna binding protein; genetics; mutation; dna-binding proteins; nonhuman; protein domain; protein motif; metabolism; protein binding; chemistry; protein multimerization; nucleotide sequence; models, molecular; mutagenesis, site-directed; protein structure, tertiary; chemical structure; helicase; protein secondary structure; enzyme structure; site directed mutagenesis; protein structure, quaternary; protein tertiary structure; oligomerization; dna helicases; virus protein; viral proteins; virus infectivity; protein quaternary structure; oligomer; protein rep68; sf3 helicase; rep proteins, adeno associated virus 2; rep proteins, adeno-associated virus 2; adeno associated virus; adeno-associated virus; adeno-associated virus - 2; dependovirus
Journal Title: PLoS Pathogens
Volume: 8
Issue: 6
ISSN: 1553-7366
Publisher: Public Library of Science  
Date Published: 2012-06-01
Start Page: e1002764
Language: English
DOI: 10.1371/journal.ppat.1002764
PROVIDER: scopus
PMCID: PMC3375335
PUBMED: 22719256
Notes: --- - "Export Date: 1 August 2012" - "Source: Scopus"
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