Conformational Changes in Active and Inactive States of Human PP2C¿ characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry

Published: May 08, 2017

Author(s)

Sharlyn J. Mazur, Elyssia S. Gallagher, Subrata Debnath, Stewart R. Durell, Kyle W. Anderson, Lisa M. Jenkins, Ettore Appella, Jeffrey W. Hudgens

Abstract

PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange–mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30–300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192–200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.
Citation: Biochemistry
Pub Type: Journals

Keywords

catalysis, catalytic domain conformational change, human pp2c¿, hydrogen/deuterium exchange-mass spectrometry, magnesium complexation, PPM phosphatase, protein phosphatase, structural dynamics
Created May 08, 2017, Updated May 15, 2017