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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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Structural comparison of SF3 helicase structures.(A) Ribbon representation of SF3 helicases AAV-2 Rep40, PV-E1 and SV40-LTag. Salmon color depicts the oligomerization domain (OD). Blue color represents the AAA+ domain. (B) PV-E1 dimer showing the residues participating in the formation of the oligomerization interface colored in red and blue. (C) Structural alignment of the OD domain of AAV-2 Rep40 (Green), BPV-E1(Blue) and SV40-LTag(Magenta).
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ppat-1002764-g001: Structural comparison of SF3 helicase structures.(A) Ribbon representation of SF3 helicases AAV-2 Rep40, PV-E1 and SV40-LTag. Salmon color depicts the oligomerization domain (OD). Blue color represents the AAA+ domain. (B) PV-E1 dimer showing the residues participating in the formation of the oligomerization interface colored in red and blue. (C) Structural alignment of the OD domain of AAV-2 Rep40 (Green), BPV-E1(Blue) and SV40-LTag(Magenta).

Mentions: As a first step in our attempt to determine the structural features that promote oligomerization in AAV Rep proteins, we analyzed the oligomeric interface of SF3 family members SV40-LTag and PV-E1. As previously described, the helicase domain contains two subdomains: a N-terminal helical bundle of four α-helices known as the oligomerization domain (OD) and the C-terminal AAA+ subdomain (Figure 1A). In PV-E1 the oligomerization interface spans both subdomains forming two extended surfaces at opposite faces of the proteins. In the AAA+ subdomain, one face comprises all the catalytic residues, including: the P-loop, its subsequent helix, the β-strands with the associated Walker B residues, sensor 1 motif, and one side of the β-hairpin (Figure 1B). The neighboring subunit interacts through areas that are located in the α-helices “behind” the β-sheet and on the opposite side of the β-hairpin (Figure 1B). Overall, about 20% of the solvent accessible area takes part in the interface and includes about 34% of all residues. In PV-E1, the OD domain consists of 68 residues forming a four helical bundle. The oligomeric interface comes from interaction of residues located in helices 1 and 4 in one monomer, with residues in helices 2, 3 and part of helix 4 in the other subunit (Figure 1B). Most of the interface is hydrophobic with many tyrosine and isoleucine residues. Similar types of interactions are seen in the interface formed by the SV40-LTag OD domains. This domain is a lot bulkier, spanning 89 residues that form a five-helix bundle. The extra helix originates from an additional Zn-finger motif. Significantly, the OD of Rep40, on the other hand, has only 52 aminoacids and, thus, is significantly shorter than PV-E1 and SV40-LTag OD domains. The direct result of this difference is a decrease in the total accessible surface area by more than 1000 Å2. In addition, the packing of the helices is less compact, producing a more dynamic structure (Figure 1C). We hypothesize that the smaller OD domain of AAV Rep proteins imparts these proteins unique oligomeric properties where the smaller Rep40/52 are mostly monomeric while Rep68/78 -with the additional OBD domain- form oligomers. However, the measurable ATPase activity in all Rep proteins, suggest that Rep40/52 should oligomerize in the presence of nucleotides [20].


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)

Structural comparison of SF3 helicase structures.(A) Ribbon representation of SF3 helicases AAV-2 Rep40, PV-E1 and SV40-LTag. Salmon color depicts the oligomerization domain (OD). Blue color represents the AAA+ domain. (B) PV-E1 dimer showing the residues participating in the formation of the oligomerization interface colored in red and blue. (C) Structural alignment of the OD domain of AAV-2 Rep40 (Green), BPV-E1(Blue) and SV40-LTag(Magenta).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375335&req=5

ppat-1002764-g001: Structural comparison of SF3 helicase structures.(A) Ribbon representation of SF3 helicases AAV-2 Rep40, PV-E1 and SV40-LTag. Salmon color depicts the oligomerization domain (OD). Blue color represents the AAA+ domain. (B) PV-E1 dimer showing the residues participating in the formation of the oligomerization interface colored in red and blue. (C) Structural alignment of the OD domain of AAV-2 Rep40 (Green), BPV-E1(Blue) and SV40-LTag(Magenta).
Mentions: As a first step in our attempt to determine the structural features that promote oligomerization in AAV Rep proteins, we analyzed the oligomeric interface of SF3 family members SV40-LTag and PV-E1. As previously described, the helicase domain contains two subdomains: a N-terminal helical bundle of four α-helices known as the oligomerization domain (OD) and the C-terminal AAA+ subdomain (Figure 1A). In PV-E1 the oligomerization interface spans both subdomains forming two extended surfaces at opposite faces of the proteins. In the AAA+ subdomain, one face comprises all the catalytic residues, including: the P-loop, its subsequent helix, the β-strands with the associated Walker B residues, sensor 1 motif, and one side of the β-hairpin (Figure 1B). The neighboring subunit interacts through areas that are located in the α-helices “behind” the β-sheet and on the opposite side of the β-hairpin (Figure 1B). Overall, about 20% of the solvent accessible area takes part in the interface and includes about 34% of all residues. In PV-E1, the OD domain consists of 68 residues forming a four helical bundle. The oligomeric interface comes from interaction of residues located in helices 1 and 4 in one monomer, with residues in helices 2, 3 and part of helix 4 in the other subunit (Figure 1B). Most of the interface is hydrophobic with many tyrosine and isoleucine residues. Similar types of interactions are seen in the interface formed by the SV40-LTag OD domains. This domain is a lot bulkier, spanning 89 residues that form a five-helix bundle. The extra helix originates from an additional Zn-finger motif. Significantly, the OD of Rep40, on the other hand, has only 52 aminoacids and, thus, is significantly shorter than PV-E1 and SV40-LTag OD domains. The direct result of this difference is a decrease in the total accessible surface area by more than 1000 Å2. In addition, the packing of the helices is less compact, producing a more dynamic structure (Figure 1C). We hypothesize that the smaller OD domain of AAV Rep proteins imparts these proteins unique oligomeric properties where the smaller Rep40/52 are mostly monomeric while Rep68/78 -with the additional OBD domain- form oligomers. However, the measurable ATPase activity in all Rep proteins, suggest that Rep40/52 should oligomerize in the presence of nucleotides [20].

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