The Distal Helix in the Regulatory Domain of Calcineurin Is Important for Domain Stability and Enzyme Function Tori B. Dunlap, Erik C. Cook, Julie Rumi-Masante, Hannah G. Arvin, Terrence E. Lester, and Trevor P. Creamer* Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone Street, Lexington, Kentucky 40536-0509, United States ABSTRACT: Calcineurin (CaN) is a calmodulin-activated, serine/threonine phosphatase that is necessary for cardiac, vasculature, and nervous system development, as well as learning and memory, skeletal muscle growth, and immune system activation. CaN is activated in a manner similar to that of the calmodulin (CaM)-activated kinases. CaM binds CaN’s regulatory domain (RD) and causes a conformational change that removes CaN’s autoinhibitory domain (AID) from its catalytic site, activating CaN. In the CaM-activated kinases, the CaM binding region (CaMBR) is located just C-terminal to the AID, whereas in CaN, the AID is 52 residues C-terminal to the CaMBR. Previously published data have shown that these 52 residues in CaN’s RD are disordered but approximately half of them gain structure, likely α-helical, upon CaM binding. In this work, we confirm that this increase in the level of structure is α-helical. We posit that this region forms an amphipathic helix upon CaM binding and folds onto the remainder of the RD:CaM complex, removing the AID. Fö rster resonance energy transfer data suggest the C-terminal end of this distal helix is relatively close to the N-terminal end of the CaMBR when the RD is bound by CaM. We show by circular dichroism spectroscopy and thermal melts that mutations on the hydrophobic face of the distal helix disrupt the structure gained upon CaM binding. Additionally, kinetic analysis of CaN activity suggests that these mutations affect CaN’s ability to bind substrate, likely a result of the AID being able to bind to the active site even when CaM is bound. Our data demonstrate the presence of this distal helix and suggest it folds onto the remainder of the RD:CaM complex, creating a hairpinlike chain reversal that removes the AID from the active site. T he calcium signaling protein calmodulin (CaM) is important in numerous signaling pathways and is known to have approximately 300 binding targets. 1 Among these targets are calmodulin-activated kinases such as CaM kinase I (CaMKI), CaM kinase II (CaMKII), CaM kinase kinase (CaMKK), and myosin light chain kinase (MLCK). 2 CaM- activated kinases possess an autoinhibitory domain (AID) that occludes the catalytic site, rendering the kinase inactive. When cellular calcium levels increase, CaM binds calcium ions and then binds the kinase at a site just C-terminal to the AID, the CaM binding region (CaMBR), causing a conformational change that removes the AID from the catalytic site, activating the kinase (Figure 1). 3 In contrast to the CaM-activated kinases, there is only one known CaM-activated phosphatase, the serine/threonine phosphatase calcineurin (CaN). 4-6 CaN is involved in several developmental processes, including formation of the cardiac, vasculature, and nervous systems. 7 CaN is also necessary for learning and memory, skeletal muscle growth, and immune system activation. 7 As such, inappropriate CaN regulation has been implicated in pathological states such as Alzheimer’s disease, 8 Down syndrome, 9 and cardiac hypertrophy. 10,11 Arguably, CaN’s most well-known target is the nuclear factor of activated T-cells (NFAT) family of transcription factors. 7 CaN dephosphorylation of NFAT reveals a cryptic nuclear localization signal that allows it to move to the nucleus and initiate a number of gene programs, including activation of T- cells. Despite its physiological importance, CaN regulation at the molecular level is still poorly understood but differs significantly from that of the CaM-activated kinases. As a result of its role in activating the immune system, CaN is the target of immunosuppressant drugs. 12 These drugs, FK506 (tacrolimus) and cyclosporin, each bind first to the prolyl isomerases FKBP12 and cyclophilin, respectively. These drug:prolyl isomerase complexes then bind CaN, inhibiting the phosphatase. 13,14 Immunosuppressant drugs are known to have numerous side effects, including serious ones such as nephrotoxicity. 15,16 There is the possibility that some side Received: April 17, 2013 Revised: October 5, 2013 Published: November 6, 2013 Figure 1. Domain ordering of CaM-activated kinases compared to the domain ordering of CaN. Article pubs.acs.org/biochemistry © 2013 American Chemical Society 8643 dx.doi.org/10.1021/bi400483a | Biochemistry 2013, 52, 8643-8651