Trapped human PPM1A-phosphopeptide complex: structural implications for regulation of PPM metal- dependent protein phosphatase activity

Published: May 25, 2018


Alexander V. Grishaev, Subrata Debnath, Dalibor Kosek, Harichandra Tagad, Stewart R. Durell, Daniel Appella, Roderico Acevedo, Fred Dyda, Ettore Appella, Sharlyn Mazur


The metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with divalent Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PP2Cα, the first PPM structure determined, displayed two tightly-bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. The structure did not provide a basis for the divalent metal ion supplementation requirement. Recent crystal structures of some bacterial or plant PPM phosphatases have displayed three metal ions in the active site. Here, the crystal structure of a trapped complex of the catalytic domain of human PPM1A with a phosphopeptide reveals three metal ions in the active site. The phosphopeptide is a cyclized variant of the activation loop of p38 MAPK, a physiological substrate of PPM1A. The PPM1A(2-297) D146E‒c(MpSIpYVA) complex provides structural confirmation of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggest that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. The position of the substrate in the active site allows solvent access to the labile third metal binding site. Furthermore, analysis of enzyme kinetics supports a random ordered bi-substrate mechanism, with substantial interaction between bound substrate and metal ions. This work provides a structural and thermodynamic basis for an innate mechanism regulating the activity of PPM phosphatases.
Citation: Journal of Biological Chemistry
Pub Type: Journals


cyclic peptide, crystallography, enzyme structure, metalloenzyme, molecular dynamics, serine/threonine phosphatase (PSP), signal transduction, small-angle X-ray scattering (SAXS)
Created May 25, 2018, Updated May 01, 2019