Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of the β12-β13 Loop in Inhibitor Binding

Abstract

Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid. The most prominent region of non-conserved sequence between these phosphatases corresponds to the ␤12-␤13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the ␤12-␤13 loop of the protein phosphatase-1 catalytic subunit (PP1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-Å resolution. The ␤12-␤13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid. Systematic mutation of each residue in the ␤12-␤13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins. Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall ␤12-␤13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.Protein phosphatase-1 (PP-1) 1 is a member of the phosphoprotein phosphatase gene family, whose members share 40% sequence identity in the catalytic subunit (PP-1c) and includes protein phosphatase-2A (PP-2A) and protein phosphatase-2B (PP-2B or calcineurin) (1). A striking structural similarity is seen upon superimposition of the crystal structures of PP-1c and the calcineurin catalytic subunit (calcineurin A), especially in the active site regions (the structure of PP-2A has not been determined). Despite the similarities in sequence and structure, PP-1c and calcineurin A have distinct inhibitor and substrate specificities. PP-1c is sensitive to the endogenous inhibitor proteins inhibitor-1 and inhibitor-2, as well as to the exogenous marine natural product inhibitors microcystin-LR (MCLR) and okadaic acid (OA). Calcineurin is markedly less sensitive to these inhibitors (250-fold for OA and over 1000-fold for the microcystins) (2-5).One region of sequence dissimilarity between PP-1c, PP2Ac, and calcineurin A is the ␤12-␤13 loop region of PP-1c/ PP-2Ac (residues 273 to 277 in PP-1c and 266 to 270 in PP-2Ac), which corresponds to the L7 loop of calcineurin A (residues 312-316). Residues of the ␤12-␤13 loop region have previously been implicated in inhibitor selectivity (6 -8). The importance of this region was first discovered when an okadaic acid-resistant isoform of PP-2A in Chinese hamster ovary cells was found to have a mutation in Cys 269 (9). The corresponding residues in PP-1c an...

Quality-control method for the determination of biological activity of engineered calcineurin subunit B

“…CNAΔ316 is a mutated form of CNA that retains the activity of the native subunit and its ability to be activated by subunit B (Wei et al, 1997;Maynes et al, 2004). The mutation increases the stability of CNA subunit, making it more suitable for quality control experiments.…”

“…CNA activity is low and this subunit is not suitable for industrial detection, storage, and transportation. We therefore used CNAΔ316 mutant, one of Loop 7 mutants, which has a higher activity and stability than native CNA and retains the characteristics of CNA interaction with CNB (Wei et al, 1997;Maynes et al, 2004). The method was developed based on the observation that CNB promotes the ability of CNAΔ316 to dephosphorylate p-nitrophenylphosphate (pNPP) (Wei et al, 1997).…”

The structural basis for Ser/Thr protein phosphatase inhibition

“…After refinement at full occupancy, the average B factor for NO is 29 Å 2 , similar to that observed for water bound to the resting state of the enzyme. The N and O atoms of NO are equidistant from the Cu, thus the Cu-nitrosyl of NiR is characterized with side-on coordination of a diatomic molecule [1].To examine the ability of the enzyme to catalyze the reverse reaction, oxidized crystals of NiR were exposed to a saturated NO solution. Refinement of the structure to 1.4 Å revealed nitrite bound to the copper via its oxygens, indicating completion of the reverse reaction in crystal.…”

“…After refinement at full occupancy, the average B factor for NO is 29 Å 2 , similar to that observed for water bound to the resting state of the enzyme. The N and O atoms of NO are equidistant from the Cu, thus the Cu-nitrosyl of NiR is characterized with side-on coordination of a diatomic molecule [1].…”

“…How this loop determines inhibitor-specificity is unknown. We have solved the structures of Protein Phosphatase-1 (PP1) bound to four marine natural product inhibitors: okadaic acid, motuporin, clavosine and microcystin-LA(2H) [1]. These inhibitors bind in a similar manner to the phosphatase, exhibiting analogous interactions and showing no structural rearrangement of the inhibitor-binding loop.…”