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.
Highlights
CN is a heterodimeric enzyme consisting of a 61-kDa subunit (CNA) with catalytic activity and the binding sites for Ca2ϩ/ CaM and a 19-kDa subunit (CNB), an EF-hand regulatory protein which binds four Ca2ϩ ions, two with very high affinity and two with more moderate affinity
Regulatory Region of Calcineurin both a detectable increase in enzyme activity and an increase in accessibility of the CaM binding segment of the CNA regulatory region, do CNB and CaM work at separate steps of CN activation, or do the two proteins work in concert to favor a single activated conformation of the enzyme? Here, we begin to address these questions using a combination of FT-IR spectroscopy, tryptophan fluorescence quenching, and functional studies of engineered CN fragments
The accepted model is that, upon activation by Ca2ϩ, CaM binds to its target sequence in CNA and induces a conformational change in CN that leads to the displacement of autoinhibitory peptide from the active site (Fig. 1)
Summary
As for the purification of CN, CN380, and CN341, cells were lysed by sonication in lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). For CaM, cells were lysed by sonication in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 mM Ca2ϩ). The reaction mixtures were titrated with 4.0 M acrylamide in the reaction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) at room temperature. FT-IR Spectra and Secondary Structure of the CN Regulatory Region—Fig. 2 shows the primary FT-IR spectrum of the isolated CN regulatory region, CNRR381–521 segment, in solution. The spectral evidence does not imply, that the isolated regulatory region has a single static conformation, but it does suggest that the region possesses some intrinsic structure
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
