Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of the beta12-beta13 Loop in Inhibitor Binding Export

@article{citeulike:3214053, 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 {beta}12-{beta}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 {beta}12-{beta}13 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-A resolution. The {beta}12-{beta}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 {beta}12-{beta}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 {beta}12-{beta}13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid. 10.1074/jbc.M407184200}, author = {Maynes, Jason T. and Perreault, Kathleen R. and Cherney, Maia M. and Luu, Hue A. and James, Michael N. and Holmes, Charles F.}, citeulike-article-id = {3214053}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M407184200}, citeulike-linkout-1 = {http://www.jbc.org/cgi/content/abstract/279/41/43198}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/15280359}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=15280359}, day = {8}, doi = {10.1074/jbc.M407184200}, journal = {J. Biol. Chem.}, keywords = {crystallography, inhibitor-2, jbc, microcystin, okadaic, phosphatase-1}, month = {October}, number = {41}, pages = {43198--43206}, posted-at = {2008-09-10 21:49:38}, priority = {0}, title = {Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of the {beta}12-{beta}13 Loop in Inhibitor Binding}, url = {http://dx.doi.org/10.1074/jbc.M407184200}, volume = {279}, year = {2004} }

TY - JOUR ID - citeulike:3214053 L3 - citeulike-article-id:3214053 N2 - 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 {beta}12-{beta}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 {beta}12-{beta}13 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-A resolution. The {beta}12-{beta}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 {beta}12-{beta}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 {beta}12-{beta}13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid. 10.1074/jbc.M407184200 IS - 41 JF - J. Biol. Chem. EP - 43206 TI - Crystal Structure and Mutagenesis of a Protein Phosphatase-1:Calcineurin Hybrid Elucidate the Role of the {beta}12-{beta}13 Loop in Inhibitor Binding VL - 279 SP - 43198 KW - crystallography KW - inhibitor-2 KW - jbc KW - microcystin KW - okadaic KW - phosphatase-1 AU - Maynes, Jason AU - Perreault, Kathleen AU - Cherney, Maia AU - Luu, Hue AU - James, Michael AU - Holmes, Charles PY - 2004/10/08/ UR - http://dx.doi.org/10.1074/jbc.M407184200 ER -