Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state

Peter G. Miller, Murugappan Sathappa, Jamie A. Moroco, Wei Jiang, Yue Qian, Sumaiya Iqbal, Qi Guo, Andrew O. Giacomelli, Subrata Shaw, Camille Vernier, Besnik Bajrami, Xiaoping Yang, Cerise Raffier, Adam S. Sperling, Christopher J. Gibson, Josephine Kahn, Cyrus Jin, Matthew Ranaghan, Alisha Caliman, Merissa Brousseau, Eric S. Fischer, Robert Lintner, Federica Piccioni, Arthur J. Campbell, David E. Root, Colin W. Garvie () and Benjamin L. Ebert ()
Additional contact information
Peter G. Miller: Harvard Medical School
Murugappan Sathappa: Broad Institute of MIT and Harvard University
Jamie A. Moroco: Broad Institute of MIT and Harvard University
Wei Jiang: Broad Institute of MIT and Harvard University
Yue Qian: Broad Institute of MIT and Harvard University
Sumaiya Iqbal: Broad Institute of MIT and Harvard University
Qi Guo: Broad Institute of MIT and Harvard University
Andrew O. Giacomelli: Broad Institute of MIT and Harvard University
Subrata Shaw: Broad Institute of MIT and Harvard University
Camille Vernier: Broad Institute of MIT and Harvard University
Besnik Bajrami: Broad Institute of MIT and Harvard University
Xiaoping Yang: Broad Institute of MIT and Harvard University
Cerise Raffier: Broad Institute of MIT and Harvard University
Adam S. Sperling: Broad Institute of MIT and Harvard University
Christopher J. Gibson: Broad Institute of MIT and Harvard University
Josephine Kahn: Harvard Medical School
Cyrus Jin: Dana-Farber Cancer Institute
Matthew Ranaghan: Broad Institute of MIT and Harvard University
Alisha Caliman: Broad Institute of MIT and Harvard University
Merissa Brousseau: Broad Institute of MIT and Harvard University
Eric S. Fischer: Dana-Farber Cancer Institute
Robert Lintner: Broad Institute of MIT and Harvard University
Federica Piccioni: Broad Institute of MIT and Harvard University
Arthur J. Campbell: Broad Institute of MIT and Harvard University
David E. Root: Broad Institute of MIT and Harvard University
Colin W. Garvie: Broad Institute of MIT and Harvard University
Benjamin L. Ebert: Broad Institute of MIT and Harvard University

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: Abstract PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.

Date: 2022
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DOI: 10.1038/s41467-022-30463-9

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