University of Massachusetts Medical School Faculty Publications
Title
Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology; Graduate School of Biomedical Sciences
Publication Date
2020-04-18
Document Type
Article Preprint
Disciplines
Amino Acids, Peptides, and Proteins | Biochemistry | Enzymes and Coenzymes | Structural Biology
Abstract
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 actively 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 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 cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted 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 auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel 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.
Keywords
biochemistry, Replication Factor C, DNA replication, sliding clamp, DNA replication, AAA+, ATPase, clamp loader
Rights and Permissions
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
DOI of Published Version
10.1101/2020.02.18.953257
Source
bioRxiv 2020.02.18.953257; doi: https://doi.org/10.1101/2020.02.18.953257. Link to preprint on bioRxiv service.
Journal/Book/Conference Title
bioRxiv
Repository Citation
Gaubitz C, Liu X, Magrino J, Stone NP, Landeck J, Hedglin M, Kelch BA. (2020). Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1101/2020.02.18.953257. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1693
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Included in
Amino Acids, Peptides, and Proteins Commons, Biochemistry Commons, Enzymes and Coenzymes Commons, Structural Biology Commons