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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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Effect of nucleotides in the oligomerization of Rep extended-linker construct proteins.ATP and ADP were added to each linker construct and compared to Rep40. Sedimentation profiles of (A) Rep40, (B) Rep68Δ219, (C) Rep68Δ214, (D) Rep68Δ209 and (E) Rep68Δ200. All protein concentrations were kept at 36 µM and contain 5 mM of ADP (left panel) or 5 mM ATP (right panel). Sedimentation velocity experiments were run at 40000 rpm and 20°C. Data was collected using the interference system.
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ppat-1002764-g005: Effect of nucleotides in the oligomerization of Rep extended-linker construct proteins.ATP and ADP were added to each linker construct and compared to Rep40. Sedimentation profiles of (A) Rep40, (B) Rep68Δ219, (C) Rep68Δ214, (D) Rep68Δ209 and (E) Rep68Δ200. All protein concentrations were kept at 36 µM and contain 5 mM of ADP (left panel) or 5 mM ATP (right panel). Sedimentation velocity experiments were run at 40000 rpm and 20°C. Data was collected using the interference system.

Mentions: In order to determine the contribution of ATP and ADP to the oligomerization of the extended linker Rep linker constructs, we performed sedimentation velocity studies in the presence of nucleotides. Our hypothesis was that if oligomerization reflects the functional state of these proteins, the addition of nucleotides should support and induce further oligomerization. Figure 5 shows that the presence of ATP and ADP induces the formation of higher order oligomers. Formation of dimeric species at this concentration can be seen with Rep68Δ214 as well as the longer constructs RepΔN209 and RepΔN200. In the later two, ADP produces two main populations sedimenting at ∼3S and ∼7S with additional intermediate oligomers. ATP on the other hand, seems to generate more stable species at ∼7S. Again, these data show that the presence of the linker region induces oligomerization of the Rep constructs and that the addition of nucleotides, in particular ATP, induces formation of larger oligomers, possibly through the stabilization of the interface formed by the AAA+ domains. This finding is in good agreement with the unique characteristics of the AAV Rep nucleotide binding pocket, which, based on its open conformation together with the presence of an arginine finger predicts the nucleotide contribution to oligomerization [24].


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)

Effect of nucleotides in the oligomerization of Rep extended-linker construct proteins.ATP and ADP were added to each linker construct and compared to Rep40. Sedimentation profiles of (A) Rep40, (B) Rep68Δ219, (C) Rep68Δ214, (D) Rep68Δ209 and (E) Rep68Δ200. All protein concentrations were kept at 36 µM and contain 5 mM of ADP (left panel) or 5 mM ATP (right panel). Sedimentation velocity experiments were run at 40000 rpm and 20°C. Data was collected using the interference system.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375335&req=5

ppat-1002764-g005: Effect of nucleotides in the oligomerization of Rep extended-linker construct proteins.ATP and ADP were added to each linker construct and compared to Rep40. Sedimentation profiles of (A) Rep40, (B) Rep68Δ219, (C) Rep68Δ214, (D) Rep68Δ209 and (E) Rep68Δ200. All protein concentrations were kept at 36 µM and contain 5 mM of ADP (left panel) or 5 mM ATP (right panel). Sedimentation velocity experiments were run at 40000 rpm and 20°C. Data was collected using the interference system.
Mentions: In order to determine the contribution of ATP and ADP to the oligomerization of the extended linker Rep linker constructs, we performed sedimentation velocity studies in the presence of nucleotides. Our hypothesis was that if oligomerization reflects the functional state of these proteins, the addition of nucleotides should support and induce further oligomerization. Figure 5 shows that the presence of ATP and ADP induces the formation of higher order oligomers. Formation of dimeric species at this concentration can be seen with Rep68Δ214 as well as the longer constructs RepΔN209 and RepΔN200. In the later two, ADP produces two main populations sedimenting at ∼3S and ∼7S with additional intermediate oligomers. ATP on the other hand, seems to generate more stable species at ∼7S. Again, these data show that the presence of the linker region induces oligomerization of the Rep constructs and that the addition of nucleotides, in particular ATP, induces formation of larger oligomers, possibly through the stabilization of the interface formed by the AAA+ domains. This finding is in good agreement with the unique characteristics of the AAV Rep nucleotide binding pocket, which, based on its open conformation together with the presence of an arginine finger predicts the nucleotide contribution to oligomerization [24].

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