Spontaneous and chaperone-assisted metal loading in the active site of protein phosphatase-1.

Abstract

Protein phosphatase PP1 has two active-site metals (Zn²⁺/Fe²⁺) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn²⁺-ions in its active site, indicating that the incorporation of Zn²⁺/Fe²⁺ depends on additional eukaryotic component(s). Here, we used purified, metal-deficient PP1 to study metal incorporation. Fe²⁺ was incorporated spontaneously, but Zn²⁺ was not. Mn²⁺-incorporation at physiological pH depended on the co-expression of PP1 with PPP1R2 (Inhibitor-2) or PPP1R11 (Inhibitor-3), or a pre-incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn²⁺-binding proteins but are, by themselves, not able to load PP1 with Zn²⁺. Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co-chaperone(s) and/or specific modification(s) for the transfer of associated Zn²⁺ to PP1.