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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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Minimum linker length requirements to induce oligomerization.We generated protein constructs with decreasing lengths of linker that are named in the context of the full Rep68 protein: comparison of the sedimentation velocity profiles of (A) Rep40, (B) Rep68ΔN219 (residues 220–536), (C) Rep68ΔN214 (residues 215–536) and (D) Rep68ΔN209 (residues 210–536). Monomeric species proteins sediment at ∼2.7S while the peak at ∼3.7S corresponds to a dimer. Protein concentration was at 36 µM in all the sedimentation experiments and run at 40000 rpm and 20°C.
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ppat-1002764-g004: Minimum linker length requirements to induce oligomerization.We generated protein constructs with decreasing lengths of linker that are named in the context of the full Rep68 protein: comparison of the sedimentation velocity profiles of (A) Rep40, (B) Rep68ΔN219 (residues 220–536), (C) Rep68ΔN214 (residues 215–536) and (D) Rep68ΔN209 (residues 210–536). Monomeric species proteins sediment at ∼2.7S while the peak at ∼3.7S corresponds to a dimer. Protein concentration was at 36 µM in all the sedimentation experiments and run at 40000 rpm and 20°C.

Mentions: Next, we sought to determine the minimal length of linker that is needed to promote oligomerization. We generated three additional constructs, named Rep68ΔN209, Rep68ΔN214 and Rep68ΔN219 and tested their ability to oligomerize (Figure 4). Our results indicate that Rep68ΔN214 contains the minimal length of linker that is required to promote detectible oligomerization, although with the shorter construct Rep68ΔN219, a small shoulder is seen at higher concentration (data not shown). These results confirm that the linker region from 215 to 224 may fold into a α-helix, resulting in an increase of the surface accessible area of the OD domain that mediates oligomerization. This increase, however, is not sufficient to produce higher order oligomers.


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)

Minimum linker length requirements to induce oligomerization.We generated protein constructs with decreasing lengths of linker that are named in the context of the full Rep68 protein: comparison of the sedimentation velocity profiles of (A) Rep40, (B) Rep68ΔN219 (residues 220–536), (C) Rep68ΔN214 (residues 215–536) and (D) Rep68ΔN209 (residues 210–536). Monomeric species proteins sediment at ∼2.7S while the peak at ∼3.7S corresponds to a dimer. Protein concentration was at 36 µM in all the sedimentation experiments and run at 40000 rpm and 20°C.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002764-g004: Minimum linker length requirements to induce oligomerization.We generated protein constructs with decreasing lengths of linker that are named in the context of the full Rep68 protein: comparison of the sedimentation velocity profiles of (A) Rep40, (B) Rep68ΔN219 (residues 220–536), (C) Rep68ΔN214 (residues 215–536) and (D) Rep68ΔN209 (residues 210–536). Monomeric species proteins sediment at ∼2.7S while the peak at ∼3.7S corresponds to a dimer. Protein concentration was at 36 µM in all the sedimentation experiments and run at 40000 rpm and 20°C.
Mentions: Next, we sought to determine the minimal length of linker that is needed to promote oligomerization. We generated three additional constructs, named Rep68ΔN209, Rep68ΔN214 and Rep68ΔN219 and tested their ability to oligomerize (Figure 4). Our results indicate that Rep68ΔN214 contains the minimal length of linker that is required to promote detectible oligomerization, although with the shorter construct Rep68ΔN219, a small shoulder is seen at higher concentration (data not shown). These results confirm that the linker region from 215 to 224 may fold into a α-helix, resulting in an increase of the surface accessible area of the OD domain that mediates oligomerization. This increase, however, is not sufficient to produce higher order oligomers.

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