The Distal Helix in the Regulatory Domain of Calcineurin Is Important for Domain Stability and Enzyme Function

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. Förster 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.

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

“…Since the AID in CN is located ~50 residues C-terminal of the CBD, and most of the C-terminal region of CNA apart from AID (457-482) is missing from previously published structures, how the binding of CaM to CBD of CN transmits through the ~50-residue linker to displace AID from the catalytic site is a topic of ongoing debate. Recently, Rumi-Masante and colleagues used CD spectroscopy, hydrogen-deuterium exchange mass spectrometry, and limited proteolytic digestions to show that the isolated RD fragment of CN is disordered but gains structure upon CaM binding [25,34]. This structure includes the expected α-helix in CBD and a so-called distal helix lying somewhere between the end of CBD and the beginning of AID.…”

“…In this model, CN activation involves multiple steps: (1) at low Ca 2+ concentration, CN exists in an inactive state in which only two high-affinity binding sites on CNB are occupied by Ca 2+ and several distinct parts of the CNA regulatory region interact with the catalytic core (the catalytic core include the CNA catalytic domain plus BBH and CNB) (Form I or the resting conformation) [13,31]; (2) in response to elevated calcium level, occupancy of the low-affinity sites on CNB by Ca 2+ induces a conformational change in CNB and triggers movement of a larger part of the CNA regulatory region, stimulating the basal activity of CN (Form II, the crystal structures of CN α and β might represent snapshots of the ensemble with different conformations) [13,33]; (3) binding of CaM to CBD, causing CBD to form an α-helix, leads to the dissociation of AIS from the LxVP-docking pocket and npg www.cell-research.com | Cell Research a conformational rearrangement of the AID segment, which results in further activation of CN (Form III) [25,34,35]; (4) CN is fully activated via a limited hydrolysis which removes the autoinhibitory regions in the C-terminus of CNA (Form IV) [36]. This new model can also be used to explain the truncation experiments and trans-inhibition data shown in Figures 3-5.…”

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

“…For simplicity, the first three residues (385–387) and the C-terminal Histidine tag residues (469–481) are numbered as part of the sequence, even though they are not part of the wild-type RD domain. The total RD construct, containing 97 residues, corresponds to constructs used previously for the study of the RD, and purification proceeded as described previously (Dunlap, Cook et al 2013, Dunlap, Guo et al 2014). Transformed cells were incubated in 100 ml terrific broth (TB; 12 g/L tryptone, 24 g/L yeast extract, 4.0 ml glycerol, 0.17 M KH 2 PO 4 , 0.72 M K 2 HPO 4 , 75 μg/ml ampicillin and 50 μg/ml kanamycin) overnight at 37°C.…”

“…In the absence of calcium-loaded CaM, the RD is disordered (Manalan and Klee 1983, Yang and Klee 2000, Rumi-Masante, Rusinga et al 2012). Binding of CaM to CaN causes a structural ordering in the RD that removes the AID from the catalytic site of CaN, thereby activating the enzyme (Shen, Li et al 2008, Dunlap, Cook et al 2013, Dunlap, Guo et al 2014, Zhao, Yang et al 2014). …”

Oxidation-Induced Conformational Changes in Calcineurin Determined by Covalent Labeling and Tandem Mass Spectrometry

“…1,7 In addition, a section of the unstructured region within the CNA subunit that is important for stabilizing the interactions within CNA upon calmodulin binding has recently been identified. 8−10 The CN active site, which is located in the catalytic domain, contains a binuclear metal center that is critical for phosphatase activity. The two metals have been identified as Zn, which is coordinated with Asn 150A , His 199A , and His 281A , (the subscript number and letter indicate the residue number and the CN subunit, respectively), and Fe, which is coordinated with Asp 90A and His 92A .…”