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

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.

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors

“…In this extended conformation, K218 and other residues involved in substrate recognition are far from the active site (i.e., the distance between K218s sidechain to D105s sidechain grows from 9 Å in the AF structure to as much as 29 Å in simulations). The two peaks seen in the flap domain to active site distance distribution are consistent with both hydrogen deuterium exchange mass spectrometry and sedimentation velocity ultracentrifugation experiments (Miller et al, 2022), which showed that PPM1D exists in an equilibrium between two different flap domain conformations.…”

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors

“…In this extended conformation, K218 and other residues involved in substrate recognition are far from the active site (i.e., the distance between K218's sidechain to D105's sidechain grows from 9 Å in the AF structure to as much as 29 Å in simulations). The two peaks seen in the flap domain to active site distance distribution are consistent with both hydrogen deuterium exchange mass spectrometry and sedimentation velocity ultracentrifugation experiments (Miller et al, 2022), which showed that PPM1D exists in an equilibrium between two different flap domain conformations.…”

“…Often, these domains are highly flexible. (Miller et al, 2022) Human protein phosphatase, Mg2+/Mn2+ dependent 1D PPM1D, also known as Wip1, is an important therapeutic target in oncology. (Pecháčková et al, 2017) PPM1D negatively regulates p53 and other components of the DNA damage response pathway.…”

Defining protein variant functions using high-complexity mutagenesis libraries and enhanced mutant detection software ASMv1.0

“…The benefits of these approaches are limited to and deemed practical to small ORFs only. Our methodology, on the other hand, emphasizes robust variant-calling tools that can process massive shotgun NGS data to detect and accurately quantify variants, and consequently allows one to screen a single saturated variant library for an ORF of any size (e.g., ~600-bp KRAS[14] , ~1200-bp TP53[15] and SMARCB1 , ~1400-bp region of PDE3A[16] , ~1700-bp SHOC2[17] , ~1800-bp PPM1D[18] , ~2200-bp EZH2 , ~3600-bp EGFR ) in a single experiment ( Fig. 1b ).…”

“…Our methodology, on the other hand, permits screening of very large pools in a single experiment using robust variant-calling tools that can accurately quantify variants from sequencing data generated by amplifying and randomly shearing the entire ORF. A single site-saturated variant library is thus adequate a wide range of ORF lengths, e.g., ~600-bp KRAS [8], ~1200-bp TP53 [9] and SMARCB1 , ~1400-bp catalytic region of PDE3A [10], ~1700-bp SHOC2 [11], ~1800-bp PPM1D [12], ~2200-bp EZH2 , ~3600-bp EGFR ) ( Fig. 1b ).…”