Abstract
Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 865-876 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 374 |
| Issue number | 4 |
| DOIs | |
| State | Published - Dec 7 2007 |
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Keywords
- OB-fold
- X-ray crystallography
- helix bundle
- replication protein A
- subunit interaction
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology
Cite this
Structure of the Full-length Human RPA14/32 Complex Gives Insights into the Mechanism of DNA Binding and Complex Formation. / Deng, Xiaoyi; Habel, Jeff E.; Kabaleeswaran, Venkataramen; Snell, Edward H.; Wold, Marc S.; Borgstahl, Gloria E.
In: Journal of Molecular Biology, Vol. 374, No. 4, 07.12.2007, p. 865-876.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Structure of the Full-length Human RPA14/32 Complex Gives Insights into the Mechanism of DNA Binding and Complex Formation
AU - Deng, Xiaoyi
AU - Habel, Jeff E.
AU - Kabaleeswaran, Venkataramen
AU - Snell, Edward H.
AU - Wold, Marc S.
AU - Borgstahl, Gloria E
PY - 2007/12/7
Y1 - 2007/12/7
N2 - Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.
AB - Replication protein A (RPA) is the ubiquitous, eukaryotic single-stranded DNA (ssDNA) binding protein and is essential for DNA replication, recombination, and repair. Here, crystal structures of the soluble RPA heterodimer, composed of the RPA14 and RPA32 subunits, have been determined for the full-length protein in multiple crystal forms. In all crystals, the electron density for the N-terminal (residues 1-42) and C-terminal (residues 175-270) regions of RPA32 is weak and of poor quality indicating that these regions are disordered and/or assume multiple positions in the crystals. Hence, the RPA32 N terminus, that is hyperphosphorylated in a cell-cycle-dependent manner and in response to DNA damaging agents, appears to be inherently disordered in the unphosphorylated state. The C-terminal, winged helix-loop-helix, protein-protein interaction domain adopts several conformations perhaps to facilitate its interaction with various proteins. Although the ordered regions of RPA14/32 resemble the previously solved protease-resistant core crystal structure, the quaternary structures between the heterodimers are quite different. Thus, the four-helix bundle quaternary assembly noted in the original core structure is unlikely to be related to the quaternary structure of the intact heterotrimer. An organic ligand binding site between subunits RPA14 and RPA32 was identified to bind dioxane. Comparison of the ssDNA binding surfaces of RPA70 with RPA14/32 showed that the lower affinity of RPA14/32 can be attributed to a shallower binding crevice with reduced positive electrostatic charge.
KW - OB-fold
KW - X-ray crystallography
KW - helix bundle
KW - replication protein A
KW - subunit interaction
UR - http://www.scopus.com/inward/record.url?scp=35748971020&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=35748971020&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2007.09.074
DO - 10.1016/j.jmb.2007.09.074
M3 - Article
VL - 374
SP - 865
EP - 876
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -