Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange–Mass Spectrometry

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.