Structural Basis for Activation of Calcineurin by Calmodulin

Abstract

The highly conserved phosphatase calcineurin 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. Calmodulin binds to a regulatory domain within calcineurin, causing a conformational change that displaces an autoinhibitory domain from the active site, resulting in activation of the phosphatase. This is the same general mechanism by which calmodulin activates calmodulin-dependent protein kinases. Previously published data has hinted that the regulatory domain of calcineurin is intrinsically disordered. In this work we demonstrate that the regulatory domain is unstructured and that it folds upon binding calmodulin, ousting the autoinhibitory domain from the catalytic site. The regulatory domain is 95 residues long, with the autoinhibitory domain attached to its C-terminal end and the 24 residue calmodulin binding region towards the N-terminal end. This is unlike the calmodulin-dependent protein kinases which have calmodulin binding sites and autoinhibitory domains immediately adjacent in sequence. Our data demonstrate that not only does the calmodulin binding region fold, but that an ~25-30 residue region between it and the autoinhibitory domain also folds, resulting in over half of the regulatory domain adopting α-helical structure. This appears to be the first observation of calmodulin inducing folding of this scale outside of its binding site on a target protein.

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

“…Together, both the trans-inhibition assay and the pull-down assay results indicate that binding of RD 398-500 to CN is not abolished when CaM binds to the CBD in RD 398-500 . This result appears to be in contradiction to the fluorescence anisotropy experiments reported by Rumi-Masante et al [25]. They found that although fl-RD 373-521 , a fluorescently labeled construct, can bind to the truncated CN 1-373 (equivalent to RD 382-524 and CN in human CN β, respectively) as indicated by an increase in anisotropy, there was no measurable anisotropy change for fl-RD 373-521 in the presence of CaM or of CaM plus CN .…”

“…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.…”

Minimizing Carry-Over in an Online Pepsin Digestion System used for the H/D Exchange Mass Spectrometric Analysis of an IgG1 Monoclonal Antibody

“…Mass analysis reveals the kinetics of deuterium incorporation, usually expressed in the form of a deuterium uptake curve. The technique is widely used to detect ligand binding [5] and to understand ligand-induced conformational changes [6], to define the effects of protein-protein interactions [7,8], to understand the effects of post-translational modifications or other chemical changes [9], to characterize the dynamics of intrinsically disordered proteins [10,11], to map protein-protein interfaces [12], and to evaluate therapeutic proteins as part of biopharmaceutical comparability studies [9].…”

Back to the Future: Nuclear Magnetic Resonance and Bioinformatics Studies on Intrinsically Disordered Proteins

“…This region has been characterized as disordered by regions of missing electron density located on both sides of the bound autoinhibitory domain as observed in the X-ray-determined structure (Kissinger et al 1995). In addition, this same region has been indicated to be disordered by protease digestion, for which multiple sites are cleaved more or less simultaneously (Manalan and Klee 1983), and also by H/D exchange with pro-teolysis coupled with mass spectrometry being used to identify the specific regions that are undergoing rapid exchange (Rumi-Masante et al 2012). To give another example, a segment within the axin scaffold protein was shown to be disordered because this segment exhibited a collapsed HSQC NMR spectrum that showed little change upon the addition of 4 M urea, because this segment failed to show evidence of unfolding upon heating, and because this segment migrated in size exclusion chromatography as a protein with a much larger mass.…”