Crystal structure and mutagenesis of a protein phosphatase-1:calcineurin hybrid elucidate the role of the beta12-beta13 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 beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the beta12-beta13 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 beta12-beta13 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 beta12-beta13 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 beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.
Canadian Institutes of Health Research, Group in Protein Structure and Function, Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Jason T
JT
Maynes
Kathleen R
KR
Perreault
Maia M
MM
Cherney
Hue Anh
HA
Luu
Michael N G
MN
James
Charles F B
CF
Holmes
D000595:Amino Acid Sequence; D019703:Calcineurin / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008387:Marine Toxins; D052998:Microcystins; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019319:Okadaic Acid / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D019319:Okadaic Acid; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D010744:Phosphoric Monoester Hydrolases; C057862:cyanoginosin LR