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J Biol Chem. 2018 May 25;293(21):7993-8008. doi: 10.1074/jbc.RA117.001213. Epub 2018 Mar 30.

A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity.

Author information

1
From the Laboratory of Cell Biology, Center for Cancer Research, NCI, and.
2
the Laboratories of Molecular Biology and.
3
Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
4
the Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850, and.
5
the National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
6
From the Laboratory of Cell Biology, Center for Cancer Research, NCI, and mazurs@mail.nih.gov.

Abstract

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

KEYWORDS:

X-ray crystallography; cyclic peptide; enzyme structure; metalloenzyme; molecular dynamics; protein serine/threonine phosphatase (PSP); signal transduction; small-angle X-ray scattering (SAXS)

PMID:
29602904
PMCID:
PMC5971455
DOI:
10.1074/jbc.RA117.001213
[Indexed for MEDLINE]
Free PMC Article

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