TY - JOUR
T1 - Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch
AU - Gaubitz, Christl
AU - Liu, Xingchen
AU - Magrino, Joseph
AU - Stone, Nicholas P.
AU - Landeck, Jacob
AU - Hedglin, Mark
AU - Kelch, Brian A.
N1 - Funding Information:
J. Hayes, and K. D. Nam and Mrs. A. Jecrois and for advice on data processing. We thank J. Andrade and Dr. B. Ueberheide for mass spectrometry sample processing and help with result interpretation. We thank Dr. S. J. Benkovic, who graciously provided the pCDF-1b vector and the pET-Duet1 vector. We thank members of the B.A.K., Royer, and Schiffer laboratories for helpful discussions. This work was funded by American Cancer Society Research Scholar Award 440685 and the National Institute of General Medical Sciences (R01-GM127776). C.G. was supported by Postdoc Mobility Fellowships 168972 and 177859 of the Swiss National Science Foundation. We thank E. Agnello and J. Pajak for critical reading of the manuscript.
Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/9/22
Y1 - 2020/9/22
N2 - DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
AB - DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
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U2 - 10.1073/pnas.2007437117
DO - 10.1073/pnas.2007437117
M3 - Article
C2 - 32907938
AN - SCOPUS:85091554578
VL - 117
SP - 23571
EP - 23580
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 38
ER -