Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone Street, Lexington, KY 40536-0509, USA.
J Mol Biol. 2012 Jan 13;415(2):307-17. doi: 10.1016/j.jmb.2011.11.008. Epub 2011 Nov 12.
The highly conserved phosphatase calcineurin (CaN) plays vital roles in numerous processes including T-cell activation, development and function of the central nervous system, and cardiac growth. It is activated by the calcium sensor calmodulin (CaM). CaM binds to a regulatory domain (RD) within CaN, causing a conformational change that displaces an autoinhibitory domain (AID) from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which CaM activates CaM-dependent protein kinases. Previously published data have hinted that the RD of CaN is intrinsically disordered. In this work, we demonstrate that the RD is unstructured and that it folds upon binding CaM, ousting the AID from the catalytic site. The RD is 95 residues long, with the AID attached to its C-terminal end and the 24-residue CaM binding region toward the N-terminal end. This is unlike the CaM-dependent protein kinases that have CaM binding sites and AIDs immediately adjacent in sequence. Our data demonstrate that not only does the CaM binding region folds but also an ∼25- to 30-residue region between it and the AID folds, resulting in over half of the RD adopting α-helical structure. This appears to be the first observation of CaM inducing folding of this scale outside of its binding site on a target protein.
高度保守的磷酸酶钙调神经磷酸酶（CaN）在许多过程中发挥着重要作用，包括 T 细胞激活、中枢神经系统的发育和功能以及心脏生长。它被钙传感器钙调蛋白（CaM）激活。CaM 与 CaN 内的调节域（RD）结合，导致构象变化，使自动抑制域（AID）从活性位点移位，从而激活磷酸酶。这与 CaM 激活钙调蛋白依赖性蛋白激酶的一般机制相同。先前发表的数据暗示 CaN 的 RD 是固有无序的。在这项工作中，我们证明 RD 是无结构的，并且在与 CaM 结合时会折叠，从而将 AID 逐出催化位点。RD 长 95 个残基，AID 附着在其 C 末端，24 个残基的 CaM 结合区位于 N 末端。这与 CaM 依赖性蛋白激酶不同，后者的 CaM 结合位点和 AID 序列紧邻。我们的数据表明，不仅 CaM 结合区折叠，而且在它和 AID 之间的约 25-30 个残基区域也折叠，导致 RD 的一半以上采用α-螺旋结构。这似乎是首次观察到 CaM 在靶蛋白的结合位点之外诱导这种规模的折叠。
