Structural basis of calcineurin activation by calmodulin Qilu Ye a,b , Yedan Feng c , Yanxia Yin c , Frédérick Faucher b , Mark A. Currie b , Mona N. Rahman b , Jin Jin a , Shanze Li c , Qun Wei c , Zongchao Jia a,b, ⁎ a College of Chemistry, Beijing Normal University, Beijing 100875, People's Republic of China b Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada c Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing Normal University, Beijing 100875, People's Republic of China abstract article info Article history: Received 12 July 2013 Accepted 30 August 2013 Available online 7 September 2013 Keywords: Activation Autoinhibition Calcineurin Calcium Calmodulin Crystallography Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regula- tion of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical con- formation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Calcineurin is the only known calcium (Ca 2+ )/calmodulin (CaM)- dependent serine/threonine protein phosphatase [1,2], which plays critical roles in many cellular and developmental processes as well as in Ca 2+ -dependent signal transduction [3–5]. Extensive studies have been conducted on the CN-NFAT (nuclear factor of activated T cells) signaling pathway and Ca 2+ -mediated signaling pathways, including the effects of these pathways on a variety of pathogenic conditions such as schizophrenia [6], Alzheimer's disease [7–9], Down's syndrome [10], cardiac hypertrophy [11–13], diabetes [14], various cancers [15–17], abnormalities in osteoclastic bone resorption [18], and glaucoma [19]. As a heterodimeric protein, calcineurin is composed of a catalytic subunit A (CnA, ~61 kDa) and a regulatory subunit B (CnB, ~19 kDa) [5,20]. CnA is further subdivided into four functional domains: a catalytic domain, a CnB binding domain (BBH), a CaM binding domain (CBD) and an autoinhibitory domain (AID) [21,22]. CBD and AID are located in the C-terminal region of CnA and are readily cleaved by proteases [21–23]. Originally, the CBD was designated as residues 389–413 while the AID was thought to encompass residues 457–482 (native calcineurin numbering is used throughout). However, a more recent study demon- strated that CBD comprises a larger domain (at least residues 389–456) and also exhibits an autoinhibitory function along with AID [24]. Based on that work, the AID has been refined (residues 389–511), and is now considered to include both the CBD in addition to sequences corre- sponding to the originally designated AID. In this work, we shall use the updated AID definition unless otherwise stated. The active site in CnA, where substrate binds and dephosphorylation takes place, is masked by AID. Although much of AID is disordered and not observed in calcineurin structures, it is generally thought that the highly flexible AID hovers near the entrance of the active site and im- pairs protein substrate access. Furthermore, it is thought that, upon CaM binding to AID, there is a conformational change and/or displace- ment of AID from its position guarding the entrance to the active center, effectively removing the steric obstacle that compromises protein substrate access and leading to full phosphatase activation. While this CaM-dependent activation model explains the biochemical and bio- physical results, little corroborating structural evidence has been pub- lished to date. Indeed, all calcineurin structures currently available were determined in the absence of CaM. In these structures, including apo calcineurin and complexes with immunosuppressant FK506 and cyclosporin, a major part of AID is highly disordered and missing in the structure, with only a short, ordered AID helix (Ser469–Arg486) Cellular Signalling 25 (2013) 2661–2667 Abbreviations: CaM, calmodulin; AID, autoinhibitory domain; CnB, calcineurin regulatory B subunit; can, calcineurin catalytic A subunit; Ca 2+ , calcium; BBH, CnB binding domain; CBD, CaM binding domain; SAXS, small angle X-ray scattering. ⁎ Corresponding author at: Department of Biomedical and Molecular Sciences, Queen's University, 18 Stuart Street, Kingston, Ontario K7L 3N6, Canada. Tel.: +1 613 533 6277; fax: +1 613 533 2497. E-mail address: jia@queensu.ca (Z. Jia). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.08.033 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig