title: The secondary structure of calcineurin regulatory region and conformational change induced by calcium/calmodulin binding.
abstract: The protein serine/threonine phosphatase calcineurin (CN) is activated by calmodulin (CaM) in response to intracellular calcium mobilization. A widely accepted model for CN activation involves displacement of the CN autoinhibitory peptide (CN(467-486)) from the active site upon binding of CaM. However, CN activation requires calcium binding both to the low affinity sites of CNB and to CaM, and previous studies did not dissect the individual contributions of CNB and CaM to displacement of the autoinhibitory peptide from the active site. In this work we have produced separate CN fragments corresponding to the CNA regulatory region (CNRR(381-521), residues 381-521), the CNA catalytic domain truncated at residue 341, and the CNA-CNB heterodimer with CNA truncated at residue 380 immediately after the CNB binding helix. We show that the separately expressed regulatory region retains its ability to inhibit CN phosphatase activity of the truncated CN341 and CN380 and that the inhibition can be reversed by calcium/CaM binding. Tryptophan fluorescence quenching measurements further indicate that the isolated regulatory region inhibits CN activity by occluding the catalytic site and that CaM binding exposes the catalytic site. The results provide new support for a model in which calcium binding to CNB enables CaM binding to the CNA regulatory region, and CaM binding then instructs an activating conformational change of the regulatory region that does not depend further on CNB. Moreover, the secondary structural content of the CNRR(381-521) was tentatively addressed by Fourier transform infrared spectroscopy. The results indicate that the secondary structure of CNRR(381-521) fragment is predominantly random coil, but with significant amount of beta-strand and alpha-helix structures.
authors.affiliation: Department of Chemistry and Institutes of Biomedical Science and Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China.
authors.forename: Xianrong
authors.initials: X
authors.lastname: Shen
authors.forename: Huiming
authors.initials: H
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Ou
authors.forename: Wenbing
authors.initials: W
authors.lastname: Tao
authors.forename: Aichun
authors.initials: A
authors.lastname: Dong
authors.forename: Jilie
authors.initials: J
authors.lastname: Kong
authors.forename: Chaoneng
authors.initials: C
authors.lastname: Ji
authors.forename: Shaoning
authors.initials: S
authors.lastname: Yu
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D019703:Calcineurin / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D020134:Catalytic Domain; D006801:Humans; D008954:Models, Biological; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010455:Peptides; D020106:Acrylamide; D014364:Tryptophan; D019703:Calcineurin; D002118:Calcium