Different roles of Loop 7 in inhibition of calcineurin.
Calcineurin (CN) is the receptor for two immunophilin-immunosuppressant complexes, Cyp-CsA and FKBP-FK506. It is a heterodimer composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). It is also inhibited by its own auto-inhibitory domain (AID). Loop 7 is a beta-hairpin within CNA that makes close contact with bound immunophilin-immunosuppressant complexes and with the AID. To investigate the role of Loop 7 in inhibition, we generated a series of deletion and substitution mutants and examined their inhibition by Cyp-CsA, FKBP-FK506 and an AID peptide. Our results demonstrate that the contacts made by Loop 7 are critical for its role in CN inhibition. Intriguingly, single residue deletions of Val314 and neighboring residues increased inhibition by FKBP-FK506 >6-fold, whereas they reduced Cyp-CsA inhibition >3-fold and abolished inhibition by the AID peptide. Most of the single substitution mutations also decreased Cyp-CsA inhibition. Loop 7 thus plays different roles in the inhibition of CN by the different inhibitors.
Wang
Hailong
H
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, PR China.
Yao
Siyan
S
Lin
Weilin
W
Du
Yanwei
Y
Xiang
Benqiong
B
He
Shuai
S
Huang
Chao
C
Wei
Qun
Q
D000818:Animals; D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D021984:Cyclophilin A / Q000737:chemistry / Q000378:metabolism; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D020104:Immunophilins / Q000737:chemistry / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D016559:Tacrolimus / Q000737:chemistry / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000378:metabolism
D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D016572:Cyclosporine; D019703:Calcineurin; D021984:Cyclophilin A; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins; D016559:Tacrolimus