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

Zarate-Perez F, Bardelli M, Burgner JW, Villamil-Jarauta M, Das K, Kekilli D, Mansilla-Soto J, Linden RM, Escalante CR - PLoS Pathog. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America.

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.

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Residue Y224 is important for oligomerization.(A) Structure-based sequence alignment of four OD domains: AAV2 Rep, Adeno-Associated virus 2 Rep; BPV-E1, Bovine papillomavirus E1 protein; REPB, Plasmid pMV158 Replication initiator protein; SV40-Tag, Simian virus 40 large T antigen. Also shown in blue the putative OD domains sequences from Snake PV1, Snake parvovirus 1 and ptMacq NS1, Point-tailed Macaque parvovirus non-structural protein 1. Highlighted in blue are the conserved aromatic residues. (B) Ribbon diagram of the structural alignment showing the aromatic residues for AAV-2 Rep (green), SV40-Tag (magenta) and BPV-E1 (Blue). (C) Sedimentation profiles of Rep68ΔN200 and Rep68ΔN200Y224A constructs. (D) Rep68 protein is compared with the mutant Rep68Y224A at different concentrations (2.5 to 10 µM). (E) ATP effect on the oligomerization of Rep68Y224A mutant. Concentration of protein was varied as in figure D from 2.5 µM to 10 µM. ATP and MgCl2 were at 5 mM.
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ppat-1002764-g008: Residue Y224 is important for oligomerization.(A) Structure-based sequence alignment of four OD domains: AAV2 Rep, Adeno-Associated virus 2 Rep; BPV-E1, Bovine papillomavirus E1 protein; REPB, Plasmid pMV158 Replication initiator protein; SV40-Tag, Simian virus 40 large T antigen. Also shown in blue the putative OD domains sequences from Snake PV1, Snake parvovirus 1 and ptMacq NS1, Point-tailed Macaque parvovirus non-structural protein 1. Highlighted in blue are the conserved aromatic residues. (B) Ribbon diagram of the structural alignment showing the aromatic residues for AAV-2 Rep (green), SV40-Tag (magenta) and BPV-E1 (Blue). (C) Sedimentation profiles of Rep68ΔN200 and Rep68ΔN200Y224A constructs. (D) Rep68 protein is compared with the mutant Rep68Y224A at different concentrations (2.5 to 10 µM). (E) ATP effect on the oligomerization of Rep68Y224A mutant. Concentration of protein was varied as in figure D from 2.5 µM to 10 µM. ATP and MgCl2 were at 5 mM.

Mentions: We generated a model of the Rep68ΔN214 construct using the X-ray structure of Rep40 (residues 225–490) and 9 residues of the linker (215–224) that were added as a helical extension to the N-terminus. The model of the α-helix was generated using Robetta [27]. Figure 8A and 8B shows the structural alignment of the OD domain of the Rep68ΔN214 model with the OD domains of PV-E1 and SV40-LTag. The alignment shows that residue Y224 superimposes with aromatic residues F313 and W270 located at the beginning of helix 1 in the OD domains of PV-E1 and SV40-LTag respectively. Analysis of the structures of both proteins reveals that these aromatic residues play a critical role in forming and stabilizing the oligomerization interface. They pack against both the N-terminal end of helix 4 of the same subunit and the C-terminus end of helix 4 of the neighboring subunit. In order to test the hypothesis that Y224 plays an equivalent role in AAV Rep proteins, we mutated it to alanine and tested its effect on the oligomerization of Rep68ΔN200. Mutation to the smaller residue alanine should have a direct effect in the oligomerization of this protein because of the significant reduction of surface exposed area. Figure 8C shows the sedimentation profile of this mutant protein showing that it completely abolishes the formation of dimers. To confirm that residue Y224 plays an important role in the oligomerization of AAV Rep proteins, we generated a Rep68Y224A mutant and compared its ability to form oligomers with respect to wild type Rep68. Analysis of the Rep68Y224A mutant reveals that at low concentration the protein is mostly found as a monomer with a sedimentation coefficient of ∼3S. At higher concentrations, we observed the appearance of multiple peaks that correspond to dimers, trimers and larger oligomers; nevertheless, the majority of the protein is present as a monomer. The presence of ATP induces a small degree of stability to the dimeric species at 5 µM and both the 5S and 11S species at 10 µM. However, the 13S complex observed with the wild type Rep68 is not formed and most of the protein is still found as a monomer (Figure 8E). These results indicate that residue Y224 is critical for the oligomerization of AAV Rep proteins.


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

Zarate-Perez F, Bardelli M, Burgner JW, Villamil-Jarauta M, Das K, Kekilli D, Mansilla-Soto J, Linden RM, Escalante CR - PLoS Pathog. (2012)

Residue Y224 is important for oligomerization.(A) Structure-based sequence alignment of four OD domains: AAV2 Rep, Adeno-Associated virus 2 Rep; BPV-E1, Bovine papillomavirus E1 protein; REPB, Plasmid pMV158 Replication initiator protein; SV40-Tag, Simian virus 40 large T antigen. Also shown in blue the putative OD domains sequences from Snake PV1, Snake parvovirus 1 and ptMacq NS1, Point-tailed Macaque parvovirus non-structural protein 1. Highlighted in blue are the conserved aromatic residues. (B) Ribbon diagram of the structural alignment showing the aromatic residues for AAV-2 Rep (green), SV40-Tag (magenta) and BPV-E1 (Blue). (C) Sedimentation profiles of Rep68ΔN200 and Rep68ΔN200Y224A constructs. (D) Rep68 protein is compared with the mutant Rep68Y224A at different concentrations (2.5 to 10 µM). (E) ATP effect on the oligomerization of Rep68Y224A mutant. Concentration of protein was varied as in figure D from 2.5 µM to 10 µM. ATP and MgCl2 were at 5 mM.
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ppat-1002764-g008: Residue Y224 is important for oligomerization.(A) Structure-based sequence alignment of four OD domains: AAV2 Rep, Adeno-Associated virus 2 Rep; BPV-E1, Bovine papillomavirus E1 protein; REPB, Plasmid pMV158 Replication initiator protein; SV40-Tag, Simian virus 40 large T antigen. Also shown in blue the putative OD domains sequences from Snake PV1, Snake parvovirus 1 and ptMacq NS1, Point-tailed Macaque parvovirus non-structural protein 1. Highlighted in blue are the conserved aromatic residues. (B) Ribbon diagram of the structural alignment showing the aromatic residues for AAV-2 Rep (green), SV40-Tag (magenta) and BPV-E1 (Blue). (C) Sedimentation profiles of Rep68ΔN200 and Rep68ΔN200Y224A constructs. (D) Rep68 protein is compared with the mutant Rep68Y224A at different concentrations (2.5 to 10 µM). (E) ATP effect on the oligomerization of Rep68Y224A mutant. Concentration of protein was varied as in figure D from 2.5 µM to 10 µM. ATP and MgCl2 were at 5 mM.
Mentions: We generated a model of the Rep68ΔN214 construct using the X-ray structure of Rep40 (residues 225–490) and 9 residues of the linker (215–224) that were added as a helical extension to the N-terminus. The model of the α-helix was generated using Robetta [27]. Figure 8A and 8B shows the structural alignment of the OD domain of the Rep68ΔN214 model with the OD domains of PV-E1 and SV40-LTag. The alignment shows that residue Y224 superimposes with aromatic residues F313 and W270 located at the beginning of helix 1 in the OD domains of PV-E1 and SV40-LTag respectively. Analysis of the structures of both proteins reveals that these aromatic residues play a critical role in forming and stabilizing the oligomerization interface. They pack against both the N-terminal end of helix 4 of the same subunit and the C-terminus end of helix 4 of the neighboring subunit. In order to test the hypothesis that Y224 plays an equivalent role in AAV Rep proteins, we mutated it to alanine and tested its effect on the oligomerization of Rep68ΔN200. Mutation to the smaller residue alanine should have a direct effect in the oligomerization of this protein because of the significant reduction of surface exposed area. Figure 8C shows the sedimentation profile of this mutant protein showing that it completely abolishes the formation of dimers. To confirm that residue Y224 plays an important role in the oligomerization of AAV Rep proteins, we generated a Rep68Y224A mutant and compared its ability to form oligomers with respect to wild type Rep68. Analysis of the Rep68Y224A mutant reveals that at low concentration the protein is mostly found as a monomer with a sedimentation coefficient of ∼3S. At higher concentrations, we observed the appearance of multiple peaks that correspond to dimers, trimers and larger oligomers; nevertheless, the majority of the protein is present as a monomer. The presence of ATP induces a small degree of stability to the dimeric species at 5 µM and both the 5S and 11S species at 10 µM. However, the 13S complex observed with the wild type Rep68 is not formed and most of the protein is still found as a monomer (Figure 8E). These results indicate that residue Y224 is critical for the oligomerization of AAV Rep proteins.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia, United States of America.

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.

Show MeSH
Related in: MedlinePlus