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

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. Protein phosphatase PP1 has two active‐site metals (Zn2+/Fe2+) that are essential for catalysis. The authors show that Fe2+ is incorporated spontaneously in bacterially expressed, metal‐deficient PP1, while Zn2+ is not incorporated spontaneously. They also identify two regulatory subunits of PP1 (PPP1R2 and PPP1R11) as likely Zn2+‐loading chaperones.