The crystal structure of the catalytic domain from the MAPK phosphatase
Pyst1 (Pyst1−CD) has been determined at 2.35 Å. The structure
adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active
site that displays a distorted geometry in the absence of its substrate with
some similarity to the dual−specificity phosphatase cdc25. Functional
characterization of Pyst1−CD indicates it is sufficient to dephosphorylate
activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1−CD
using the substrate p−nitrophenyl phosphate (pNPP) reveals that
both molecules undergo catalytic activation in the presence of recombinant
inactive ERK2, switching from a low− to high−activity form. Mutation
of Asp 262, located 5.5 Å distal to the active site, demonstrates it
is essential for catalysis in the high−activity ERK2−dependent
conformation of Pyst1 but not for the low−activity ERK2−independent
form, suggesting that ERK2 induces closure of the Asp 262 loop over the active
site, thereby enhancing Pyst1 catalytic efficiency.
