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Published February 19, 2016 | Supplemental Material
Journal Article Open

2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition


p97 is a hexameric AAA ATPase that is an attractive target for cancer drug development. Here, we report cryo-EM structures for ADP-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 Å and 2.4 Å, respectively. We also report cryo-EM structures at ~ 3.3 Å, 3.2 Å and 3.3 Å resolutions respectively, for three distinct, co-existing functional states of p97 with occupancies of 0, 1 or 2 molecules of ATPγS per protomer. A large corkscrew-like change in molecular architecture coupled with upward displacement of the N-domain is observed only when ATPγS is bound to both D1 and D2 domains. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Additional Information

© 2016 American Association for the Advancement of Science. 3 November 2015; accepted 14 January 2016 Published online 28 January 2016. First release: 28 January 2016. This research was supported by funds from the Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, and with federal funds from the National Cancer Institute, National Institutes of Health, under Chemical Biology Consortium Contract No. HHSN261200800001E Agreement No. 29XS127TO15. RJD is an investigator of the HHMI. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We thank K. Moynihan, R. Mueller, and J. Cometa for technical assistance with electron microscopy, F. Ulmer, P. Mooney and C. Booth for advice and assistance with optimizing K2 detector performance, and M.G. Laporte for contributions to the medicinal chemistry program. The University of Pittsburgh has filed a provisional patent application that covers the inhibitor compound described in this manuscript. RD is on the Scientific Advisory Board of Cleave Biosciences. The density maps and refined atomic models have been deposited with the Electron Microscopy Data Bank with accession numbers of EMD-3295, 3296, 3297, 3298 and 3299 and the Protein Data Bank with matching accession numbers of PDB-5FTJ, 5FTK, 5FTL, 5FTM and 5FTN, respectively for native p97 in the presence and absence of bound inhibitor, and for p97 conformational states I, II and III observed in the presence of ATPγS.

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