Structural Basis for Regulation of Protein Phosphatase 1 by Inhibitor-2

Abstract

The functional specificity of type 1 protein phosphatases (PP1) depends on the associated regulatory/targeting and inhibitory subunits. To gain insights into the mechanism of PP1 regulation by inhibitor-2, an ancient and intrinsically disordered regulator, we solved the crystal structure of the complex to 2.5Å resolution. Our studies show that, when complexed with PP1c, I-2 acquires three regions of order: site 1, residues 12-17, binds adjacent to a region recognized by many PP1 regulators; site 2, amino acids 44 -56, interacts along the RVXF binding groove through an unsuspected sequence, KSQKW; and site 3, residues 130 -169, forms ␣-helical regions that lie across the substratebinding cleft. Specifically, residues 148 -151 interact at the catalytic center, displacing essential metal ions, accounting for both rapid inhibition and slower inactivation of PP1c. Thus, our structure provides novel insights into the mechanism of PP1 inhibition and subsequent reactivation, has broad implications for the physiological regulation of PP1, and highlights common inhibitory interactions among phosphoprotein phosphatase family members.

Calcium‐induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain

“…Such bi-partite binding has been described for Ste5, Oct1 [15], and bacterial cellulose [28]. Tripartite binding of an IDP has only been described for I2 binding to PP1 [16], and its general occurrence may be limited by the large and unfavorable decrease in configurational entropy, to be compensated by advantages of excess specificity.…”

“…Most often, IDPs only bind their partner via a short recognition element [14], and less frequently by virtue of two binding elements separated by a disordered linker region [15]. Only in one case so far has the tripartite binding of an IDP been described: protein phosphatase 1 (PP1) by inhibitor-2 (I2) [9,16].…”

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

“…NMDA receptor signaling leads to I-2 dephosphorylation at T72 and increases PP1-I-2 interaction. Based on in vitro biochemical studies, unphosphorylated I-2 at T72 is thought to inhibit PP1 by blocking the PP1 active site via the -helix structure on I-2 (Hurley et al, 2007), but in a delayed manner (t 1/2 30 min; Cohen, 1989). It is known that LTD stimulus only results in transient PP1 activation (45 min; Thiels et al, 1998).…”

“…Based on the crystal structure of the PP1-I-2 complex and other mostly in vitro biochemical studies, PP1 binds tightly to I-2 in a 1:1 stoichiometry, making multiple contacts with different parts of PP1, including an -helix of I-2 that covers the active site of PP1, inhibiting it (Huang et al, 1999;Hurley et al, 2007;Dancheck et al, 2011). PP1 is inactive within the in vitro PP1-I-2 complex, but can be quickly activated when I-2 is phosphorylated at threonine 72 (pT72) by GSK3 (Cohen, 1989), presumably removing the I-2 -helix away from the active site of PP1.…”

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

“…12 As an innately flexible protein, inhibitor-2 exerts its inhibitory effect through a combination of induced conformational changes and direct occlusion of the PP1 active site. 13,14 Microcystin and okadaic acid however, are two representatives of a chemically diverse and naturally occurring group of small molecule toxins that target the PPP-family enzymes. Microcystins, produced by cyanobacteria (i.e., Microcystis aeruginosa) and okadaic acid, made by dinoflagellates (i.e., Dinophysis sp), each exert their inhibitory effects by directly binding to the active site phosphatases, establishing that two SLP phosphatase isoforms exist in plants and that they maintain chloroplastic (SLP1) and cytosolic (SLP2) subcellular localizations, respectively.…”