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

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 β12-β13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the β12-β13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) 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 β12-β13 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 β12-β13 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 β12-β13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid. 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 β12-β13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the β12-β13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) 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 β12-β13 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 β12-β13 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 β12-β13 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) 1The abbreviations used are: PP-1, protein phosphatase-1; MCLR, microcystin-LR; OA, okadaic acid. 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) (1Cohen P. Bioessays. 1994; 16: 583-588Crossref PubMed Scopus (34) Google Scholar). 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) (2Sheppeck 2nd, J.E. Gauss C.M. Chamberlin A.R. Bioorg. Med. Chem. 1997; 5: 1739-1750Crossref PubMed Scopus (142) Google Scholar, 3Dawson J.F. Holmes C.F. Front. Biosci. 1999; 4: D646-D658Crossref PubMed Google Scholar, 4Bialojan C. Takai A. Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1518) Google Scholar, 5MacKintosh C. MacKintosh R.W. Trends Biochem. Sci. 1994; 19: 444-448Abstract Full Text PDF PubMed Scopus (239) Google Scholar). One region of sequence dissimilarity between PP-1c, PP-2Ac, and calcineurin A is the β12-β13 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 β12-β13 loop region have previously been implicated in inhibitor selectivity (6Watanabe T. Huang H.B. Horiuchi A. da Cruze Silva E.F. Hsieh-Wilson L. Allen P.B. Shenolikar S. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3080-3085Crossref PubMed Scopus (60) Google Scholar, 7Connor J.H. Kleeman T. Barik S. Honkanen R.E. Shenolikar S. J. Biol. Chem. 1999; 274: 22366-22372Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 8Yang J. Hurley T.D. DePaoli-Roach A.A. J. Biol. Chem. 2000; 275: 22635-22644Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 Cys269 (9Shima H. Tohda H. Aonuma S. Nakayasu M. DePaoli-Roach A.A. Sugimura T. Nagao M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9267-9271Crossref PubMed Scopus (47) Google Scholar). The corresponding residues in PP-1c and calcineurin A are Phe276 and Tyr315. Because PP-2Ac is much more sensitive to okadaic acid (IC50 = 0.2 nm) than PP-1c (IC50 = 20-50 nm) or calcineurin (IC50 = 5 μm), the presence of a cysteine at this position may be responsible for its higher sensitivity to okadaic acid. Further mutagenesis studies were carried out in PP-1c that confirmed the importance of the β12-β13 loop region in inhibitor sensitivity, as well as in enzyme activity (6Watanabe T. Huang H.B. Horiuchi A. da Cruze Silva E.F. Hsieh-Wilson L. Allen P.B. Shenolikar S. Greengard P. Nairn A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3080-3085Crossref PubMed Scopus (60) Google Scholar, 10Zhang Z. Zhao S. Long F. Zhang L. Bai G. Shima H. Nagao M. Lee E.Y. J. Biol. Chem. 1994; 269: 16997-17000Abstract Full Text PDF PubMed Google Scholar, 11Zhang L. Zhang Z. Long F. Lee E.Y. Biochemistry. 1996; 35: 1606-1611Crossref PubMed Scopus (68) Google Scholar, 12Wei Q. Lee E.Y. Biochemistry. 1997; 36: 7418-7424Crossref PubMed Scopus (27) Google Scholar). Structural studies have shed light on the nature and importance of the β12-β13 loop in PP-1c in the context of inhibitor binding. The hydroxyl of Tyr272 in the β12-β13 loop was analyzed in four different crystals of the same PP-1c·tungstate complex and found to have a metal to hydroxyl contact distance ranging from 3.0 to 4.3 Å, suggesting flexibility in the loop region (13Egloff M.P. Cohen P.T. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (380) Google Scholar). Flexibility is also noted in differences between the structures of PP-1c bound to MCLR, okadaic acid, tungstate, and calyculin A (13Egloff M.P. Cohen P.T. Reinemer P. Barford D. J. Mol. Biol. 1995; 254: 942-959Crossref PubMed Scopus (380) Google Scholar, 14Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (755) Google Scholar, 15Maynes J.T. Bateman K.S. Cherney M.M. Das A.K. Luu H.A. Holmes C.F. James M.N. J. Biol. Chem. 2001; 276: 44078-44082Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Kita A. Matsunaga S. Takai A. Kataiwa H. Wakimoto T. Fusetani N. Isobe M. Miki K. Structure (Lond.). 2002; 10: 715-724Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The loop in the MCLR-bound structure folds back on itself, shifting the β12-β13 loop relative to the tungstate-, OA-, and calyculin-bound PP-1c structures (15Maynes J.T. Bateman K.S. Cherney M.M. Das A.K. Luu H.A. Holmes C.F. James M.N. J. Biol. Chem. 2001; 276: 44078-44082Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These differences may be indicative of the covalent bond formed between MCLR and Cys273 (PP-1c), which is not needed for inhibition (17Craig M. Luu H.A. McCready T.L. Williams D. Andersen R.J. Holmes C.F. Biochem. Cell Biol. 1996; 74: 569-578Crossref PubMed Scopus (135) Google Scholar, 18MacKintosh R.W. Dalby K.N. Campbell D.G. Cohen P.T. Cohen P. MacKintosh C. FEBS Lett. 1995; 371: 236-240Crossref PubMed Scopus (240) Google Scholar, 19Holmes C.F. Maynes J.T. Perreault K.R. Dawson J.F. James M.N. Curr. Med. Chem. 2002; 9: 1981-1989Crossref PubMed Scopus (34) Google Scholar). Before the structure of OA-bound to PP-1c had been determined, it had been hypothesized that the different conformations of the L7 loop in calcineurin and the corresponding β12-β13 loop in PP-1c were the reason for the resistance of calcineurin to toxins (17Craig M. Luu H.A. McCready T.L. Williams D. Andersen R.J. Holmes C.F. Biochem. Cell Biol. 1996; 74: 569-578Crossref PubMed Scopus (135) Google Scholar, 20Dawson J.F. Luu H.A. Bagu J.R. Holmes C.F. Biochem. Biophys. Res. Commun. 2000; 270: 543-549Crossref PubMed Scopus (7) Google Scholar). However, upon superimposition of the PP-1c-OA and calcineurin A structures, it was noted that the backbone atoms of the two loops superimpose. This led to our hypothesis that the resistance of calcineurin to toxins was mainly because of primary sequence differences in the C-terminal end of the L7 loop of calcineurin and the β12-β13 loop of PP-1c/PP-2Ac (19Holmes C.F. Maynes J.T. Perreault K.R. Dawson J.F. James M.N. Curr. Med. Chem. 2002; 9: 1981-1989Crossref PubMed Scopus (34) Google Scholar). This solidifies the idea that the differences seen in the MCLR-bound PP1-c structure are not typical and are caused by the covalent reaction (14Goldberg J. Huang H.B. Kwon Y.G. Greengard P. Nairn A.C. Kuriyan J. Nature. 1995; 376: 745-753Crossref PubMed Scopus (755) Google Scholar). To test our hypothesis concerning the importance of the primary sequence on inhibitor potential, we created a chimeric mutant of PP-1c in which the β12-β13 loop residues of PP-1c (273CGEFD277) were substituted with the L7 loop residues of calcineurin A (312LDVYN316). We analyzed this PP-1c-CaN mutant via crystallographic and kinetic studies to probe further the reason for PP1-c sensitivity and the resistance to toxins and inhibitor proteins for calcineurin. Expression and Purification of Recombinant PP-1c—Mutants of PP-1cγ were expressed and purified to homogeneity as in Refs. 20Dawson J.F. Luu H.A. Bagu J.R. Holmes C.F. Biochem. Biophys. Res. Commun. 2000; 270: 543-549Crossref PubMed Scopus (7) Google Scholar and 21McCready T.L. Islam B.F. Schmitz F.J. Luu H.A. Dawson J.F. Holmes C.F. J. Biol. Chem. 2000; 275: 4192-4198Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar. The catalytic subunit of the human wild type and mutant PP-1c was expressed in Escherichia coli DH5α strain using the plasmid pCW and subsequently purified to homogeneity using heparin affinity, Mono Q, and Superdex 75 gel filtration chromatography as previously described (20Dawson J.F. Luu H.A. Bagu J.R. Holmes C.F. Biochem. Biophys. Res. Commun. 2000; 270: 543-549Crossref PubMed Scopus (7) Google Scholar, 22Alessi D.R. Street A.J. Cohen P. Cohen P.T.W. Eur. J. Biochem. 1993; 213: 1055-1066Crossref PubMed Scopus (167) Google Scholar, 23Zhang Z.J. Bai G. Deanszirattu S. Browner M.F. Lee E.Y.C. J. Biol. Chem. 1992; 267: 1484-1490Abstract Full Text PDF PubMed Google Scholar) with the following modifications. A single colony of E. coli DH5α cells transformed with plasmid pCW-HPP-1γ was used to inoculate 100 ml of Luria-Bertani media containing 1 mm MnCl2 and ampicillin (54 μm). After overnight growth at 37 °C, this culture was used to inoculate 1 liter of Luria-Bertani media containing 1 mm MnCl2 and ampicillin (54 μm) and grown to optical density 0.5 at 600 nm. Expression was then induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for up to 18 h. Cells were harvested by centrifugation for 45 min at 4,000 × and at or used cells from two were in ml of A mm 0.5 mm 0.5 mm mm 0.5 mm mm 3.0 mm and containing 100 mm and for min at × The was a PP-1c was in A by a of of was determined by the were and to in mm 0.5 mm 0.5 mm 3.0 mm mm mm 3.0 mm and The were then a Mono and in by a of mm were and to a in a a Superdex 75 in mm mm mm mm mm 3.0 mm The PP-1c was by an of by were and to in a of was and the at The of the wild type and mutant were using the a and found to between and a was used as a phosphatase substrate to with microcystin-LR, okadaic acid, and inhibitor-2. PP-1c were in phosphatase mm mm 1 mm and was a of enzyme caused in a (20Dawson J.F. Luu H.A. Bagu J.R. Holmes C.F. Biochem. Biophys. Res. Commun. 2000; 270: 543-549Crossref PubMed Scopus (7) Google Scholar, 21McCready T.L. Islam B.F. Schmitz F.J. Luu H.A. Dawson J.F. Holmes C.F. J. Biol. Chem. 2000; 275: 4192-4198Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). were for each and inhibitor less than was seen between was from okadaic acid from and was purified from E. The loop mutant protein phosphatase-1 activity a as a of were by the at The enzyme and okadaic acid were in a with the of protein The complex was then with an of which of 100 mm and mm The complex in the with a = = Å, = Å, with complex Structure and data to was at 100 on a with a The data were with the of M. 1997; 276: PubMed Scopus Google Scholar). This structure was by with the J. P. 1997; 276: PubMed Scopus Google using the PP-1c-OA structure OA as a density for the protein and the inhibitor were from the from the OA was to the density using the structure of OA bound to wild type PP-1c as a The was to in to the using the Biol. 1999; Scopus Google Scholar, P. R.W. J. M. R.J. T. D. PubMed Scopus Google Scholar). The was then to of using with a The crystallographic data are in The of density for residues and was for using and R.W. G. C. Nature. 1996; PubMed Scopus Google Scholar, J. Mol. Biol. 1993; PubMed Scopus Google Scholar). showed that of residues were in with an overall of and in to of of in to in to in to in to is the and is the from of atoms acid are the and structure was as for with of the data from structural in to is the and is the from of are the and structure was as for with of the data from structural in a and structure have been in the Protein with and of and the of the β12-β13 loop region of PP-1c in inhibitor selectivity we the of natural product inhibitors and an endogenous inhibitor protein on a mutant of PP-1c in the β12-β13 loop region This mutant to as the has residues 273-277 of PP-1c substituted with the corresponding residues of calcineurin A (residues This loop mutant has to the of this non-conserved of the type and (calcineurin) phosphatases in inhibitor in a PP-1c was by okadaic acid with an of and microcystin-LR with an of nm. The loop mutant was less sensitive to okadaic acid (IC50 = nm) and at less sensitive to microcystin-LR (IC50 = PP-1c with an of nm. The loop mutant was less sensitive to inhibition (IC50 = The resistance of the loop mutant of PP-1c to inhibitors is with the hypothesis that the residues of the β12-β13 loop region of PP-1c a in inhibitor Structure of structure of the loop mutant PP-1c bound to okadaic acid is to PP-1c structures to wild-type to MCLR in the to in the to in the over The of OA in the active site is from that seen with the wild-type with the OA a conformation via an bond between the and the between the enzyme and the inhibitor are seen in the wild-type and loop mutant this the of OA the of PP-1c created by residues and and interactions between Tyr272 and the and and the and and the The that complex is by this structure because are interactions the of the and the of these interactions is on a with the interactions seen in the wild-type acid complex (15Maynes J.T. Bateman K.S. Cherney M.M. Das A.K. Luu H.A. Holmes C.F. James M.N. J. Biol. Chem. 2001; 276: 44078-44082Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The residue the β12-β13 loop substitution of the sequence with from calcineurin A. Because the of the backbone atoms in the wild-type and the loop mutant the this as for inhibitor these may be to overall in the β12-β13 loop of the chimeric the two most prominent residues that have inhibitor interactions are and The a of OA a of the to the position of the residue in wild-type PP-1c the same of OA, this The density for Phe276 is well in wild-type it has an of the for the In the loop mutant the density of is with an of the for the of the four structures of calcineurin have for the residue that are the for the of the The in this position not have the for interactions that the in wild-type PP-1c The that inhibitor in the phosphoprotein phosphatase may of the of the inhibitors and also a to the active site residue by Residues of the β12-β13 kinetic data that the L7 loop of calcineurin A and β12-β13 loop of PP-1c inhibitor this are residues that this Tyr272 has the for because it the most with the with the acid of OA, the of calyculin and the in Tyr272 is also important in of calyculin A and because the mutation a and decrease in sensitivity to these toxins L. Zhang Z. Long F. Lee E.Y. Biochemistry. 1996; 35: 1606-1611Crossref PubMed Scopus (68) Google Scholar). This residue as a in PP-1c and calcineurin A. To the individual of the residues the β12-β13 loop inhibitor we to and the single of residues 273-277 from PP-1c to their corresponding residues in calcineurin A. to okadaic acid inhibition A and PP-1c and were by this as as the wild-type enzyme (IC50 = In a single PP-1c (IC50 = showed resistance to inhibition over wild This resistance was to that by the PP-1c loop mutant (IC50 = to microcystin-LR inhibition A and PP-1c and mutant were by this as as the wild-type enzyme (IC50 = In the single mutant PP-1c (IC50 = nm) was as to inhibition as the PP-1c loop mutant (IC50 = inhibition of PP-1c the PP-1c mutant was not to inhibition by and this as well as PP-1c and mutant was as as the wild-type enzyme (IC50 = the PP-1c loop mutant showed resistance to inhibition by (IC50 = Protein phosphatase-1 and protein phosphatase-2B (calcineurin) share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by endogenous such as protein phosphatase inhibitor-2, and natural product toxins, such as microcystin-LR and okadaic acid. One of the more prominent regions of non-conserved sequence between these phosphatases corresponds to the β12-β13 loop of protein phosphatase-1, and the L7 loop of calcineurin. In the present study, mutagenesis of residues 273-277 of the β12-β13 loop of protein phosphatase-1 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 inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-Å resolution. The of this structure was to the differences in the β12-β13 loop structure between wild-type PP-1c and the loop mutant PP-1c, bound to This was in the context of the structural seen in the complex that may have implicated structural in the loop as indicative of inhibitor However, the backbone conformation of the loop the active is in the wild-type complex and the loop mutant in a the loop has an in the tungstate-, OA-, and calyculin-bound the L7 loop of calcineurin also has a conformation to these The is the MCLR structure the covalent to Cys273 may the loop This the the that primary sequence, than the overall β12-β13 loop structure, the inhibitor A change in with of the β12-β13 loop region was by J.H. Kleeman T. Barik S. Honkanen R.E. Shenolikar S. J. Biol. Chem. 1999; 274: 22366-22372Abstract Full Text Full Text PDF PubMed Scopus (81) Google when substituted the of PP-1c (residues with the corresponding of PP-2Ac J.H. Kleeman T. Barik S. Honkanen R.E. Shenolikar S. J. Biol. Chem. 1999; 274: 22366-22372Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The chimeric protein was not by or inhibitor-1 of which the chimeric protein had the of with to and suggesting that the β12-β13 loop region is less important for inhibition by these natural We to residues in the β12-β13 loop region of PP-1c to their corresponding residues in calcineurin A. The created were PP-1c and We not Tyr272 as this residue is a in calcineurin A and our structural data showed that it an conformation in the loop mutant complex The to single in the β12-β13 loop region of PP-1c was following the of a density for in the loop mutant crystal The corresponding residue in PP-2Ac, has been found to be important for okadaic acid of Phe276 to in PP-1c the sensitivity of PP-1c to okadaic acid that an residue at this position may of okadaic acid Z. Zhao S. Long F. Zhang L. Bai G. Shima H. Nagao M. Lee E.Y. J. Biol. Chem. 1994; 269: 16997-17000Abstract Full Text PDF PubMed Google Scholar). Systematic mutation of each residue in the β12-β13 loop of PP-1c to their corresponding calcineurin A residue showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to the inhibitors microcystin-LR and okadaic acid. Taken together, these data indicate that an individual amino acid residue substitution as in calcineurin) and not a change in the overall β12-β13 loop conformation of PP-1c contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid. MacKintosh R.W. Dalby K.N. Campbell D.G. Cohen P.T. Cohen P. MacKintosh C. FEBS Lett. 1995; 371: 236-240Crossref PubMed Scopus (240) Google Scholar) first showed that Cys273 to microcystin-LR and determined that of covalent mutation to or the for inhibition of PP-1c by This is with our of the β12-β13 loop However, we have also that covalent of Cys273 by microcystin-LR over and the phosphatase is by the (17Craig M. Luu H.A. McCready T.L. Williams D. Andersen R.J. Holmes C.F. Biochem. Cell Biol. 1996; 74: 569-578Crossref PubMed Scopus (135) Google Scholar, 19Holmes C.F. Maynes J.T. Perreault K.R. Dawson J.F. James M.N. Curr. Med. Chem. 2002; 9: 1981-1989Crossref PubMed Scopus (34) Google Scholar). Taken together, the of these studies that covalent of Cys273 is with to inhibition of the phosphatase and that this residue a in the inhibition of PP-1c by the toxins (19Holmes C.F. Maynes J.T. Perreault K.R. Dawson J.F. James M.N. Curr. Med. Chem. 2002; 9: 1981-1989Crossref PubMed Scopus (34) Google Scholar). with its important in Cys273 is in members of the PP-1c A PP-1c from Silva Biochem. J. PubMed Google it is to The sensitivity to inhibition of this of PP-1c was as less than a containing Cys269 (the residue in this is that is more for inhibition in this position than and MacKintosh R.W. Dalby K.N. Campbell D.G. Cohen P.T. Cohen P. MacKintosh C. FEBS Lett. 1995; 371: 236-240Crossref PubMed Scopus (240) Google these were on protein from E. coli PP-1c from and have Cys273 to R.W. G. D.G. Cohen P.T. FEBS Lett. 276: PubMed Scopus Google Scholar, Q. J. Mol. Biol. PubMed Scopus Google Scholar, A. Mol. Biol. 1995; PubMed Scopus Google Scholar). of PP-1c were to be as sensitive to inhibitors as PP-1c C. S. Cohen P. FEBS Lett. PubMed Scopus Google Scholar, C. Cohen P. Biochem. J. PubMed Scopus Google it be to of purified PP-1c from these have resistance to In to the of Cys273 in mediating inhibition of PP-1c, of Cys273 with not inhibition of PP-1c by inhibitor-2. The substitution that inhibition was that the of inhibition of PP-1c by this endogenous inhibitor protein is distinct from that of the natural The of in the β12-β13 loop of PP-1c on inhibition by are to because the structure of this protein bound to PP-1c is The differences in sensitivity to the inhibitor protein between wild-type PP-1c and calcineurin A are much more than the differences between wild-type PP-1c and loop mutant PP-1c, that the β12-β13 loop of PP-1c is not the region a in of this Taken together, these data are with the most of J. Hurley T.D. DePaoli-Roach A.A. J. Biol. Chem. 2000; 275: 22635-22644Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The importance of the β12-β13 loop was in a structure of the complex between PP-1c and a from phosphatase subunit M. F. K. T. Nature. PubMed Scopus Google Scholar). This structure showed active site with subunit to the catalytic subunit the β12-β13 loop in a prominent position to activity and of the of the β12-β13 loop of PP-1c in mediating inhibition of this enzyme by toxins protein of calcineurin A that is sensitive to This be an first in active inhibitors that be for calcineurin. We Barford for the of the PP-1c·tungstate

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