In a recent issue of Biochemical Journal, Brohus et al. (Biochem. J.476, 193–209) investigated the interaction between the ubiquitous intracellular Ca2+-sensor calmodulin (CaM) and peptides that mimic different structural regions of the cardiac ryanodine receptor (RyR2) at different Ca2+ concentrations. For the purpose, a novel bidimensional titration assay based on changes in fluorescence anisotropy was designed. The study identified the CaM domains that selectively bind to a specific CaM-binding domain in RyR2 and demonstrated that the interaction occurs essentially under Ca2+-saturating conditions. This study provides an elegant and experimentally accessible framework for detailed molecular investigations of the emerging life-threatening arrhythmia diseases associated with mutations in the genes encoding CaM. Furthermore, by allowing the measurement of the equilibrium dissociation constant in a protein–protein complex as a function of [Ca2+], the methodology presented by Brohus et al. may have broad applicability to the study of Ca2+ signalling.
The crucial role of Ca2+ as a universal second messenger is largely established and the variety of biochemical pathways directly or indirectly regulated by Ca2+ has been extensively investigated. In more recent years, the important contribution of Ca2+ signalling in cardiac myocytes has become increasingly clear. Growing evidence has been collected as to its direct involvement in the regulation of action potentials and arrhythmias as well as in the modulation of contractile function, transcription regulation and mitochondrial function . The ubiquitous Ca2+ sensor calmodulin (CaM) directly participates in the modulation of the cardiac action potential by binding to and controlling the modulation of the voltage-gated potassium, sodium and calcium channels . CaM is a relatively small (148 amino acids) α-helical protein composed of distinct N- and C-terminal domains separated by a flexible linker region. Each domain contains two Ca2+ binding EF-hand motifs (Figure 1) that bind Ca2+ with a significantly different affinity. Indeed, in the absence of the target, the C-domain binds the cation much stronger (apparent Kd ∼ 1 μM) than the N-domain (apparent Kd ∼ 10 μM) . Positive cooperativity of Ca2+ binding occurs within each domain, however, in the absence of the target no cooperativity is detected between the N- and C-domains, which bind Ca2+ independently of each other . The high specificity and selectivity of CaM-mediated target activation is the consequence of variations in the number of Ca2+ ions required to bind CaM for target activation  and of the free energy coupling between Ca2+ binding and target binding, which results in changes of cooperativity of Ca2+ binding of the CaM-target complex [5,6]. To properly regulate the specific biological process, a tripartite equilibrium between Ca2+, CaM and its target is, therefore, established, which relies on microscopic equilibria that may influence each other in a rather complex fashion.
In cardiomyocytes, the release of Ca2+ from the sarcoplasmic reticulum is controlled by the large intracellular Ca2+ channel referred to as ryanodine receptor 2 (RyR2) . The complex homo-tetrameric arrangement of the RyR2 channel has been recently solved by cryogenic electron microscopy (cryoEM) [8–10], offering an unprecedented insight into the mechanism known as Ca2+-induced Ca2+ release (CICR). This phenomenon follows the activation of RyR2 caused by the rise in cytosolic Ca2+ upon excitation of cardiomyocytes, and it is necessary to cause myofilament contraction . CaM modulates the activity of RyR2 by inhibiting the release of Ca2+ in a Ca2+-dependent manner, a mechanism that is achieved by selective binding to specific regions of the channel with a 1 : 1 stoichiometry, during [Ca2+] oscillations in the ∼0.2 to 200–400 μM range (at diastole and during CICR and systole, respectively) [11,12]. In particular, it has been established that apo-CaM has either no effect or inhibitory effect on RyR2, while Ca2+-CaM inhibits the channel .
The full characterization of the mechanisms underlying the Ca2+-dependent binding of CaM to RyR2 is, therefore, crucial to achieve a complete molecular understanding of processes occurring in cardiomyocytes. This is especially apparent in light of the relatively recent discovery that mutations in the genes encoding CaM are associated with ventricular tachycardia and sudden cardiac death . To date, eighteen point mutations in either CALM1, CALM2 or CALM3, all genes independently encoding CaM in the human genome, have been associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome or idiopathic ventricular fibrillation [15,16] (Figure 1). This surprising discovery contrasting with the high level of evolutionary conservation of CaM led to the definition a new branch of pathology named calmodulinopathy, with specific clinical phenotypes affecting mostly young individuals .
In a recent work published in the Biochemical Journal, Brohus et al.  unveiled the molecular details of CaM-RyR2 interaction by identifying the CaM-binding domain in RyR2 at varying calcium concentration ([Ca2+]). This is not a trivial task, as the affinity of CaM for Ca2+ has been shown to greatly depend on the target, often significantly increasing in its presence [19–21]. Moreover, the binding of CaM to its target is itself governed by a specific equilibrium, which needs to be investigated independently and can also be influenced by the presence of Ca2+. Therefore, one normally needs to perform a long series of titration experiments, in which either the protein concentration is kept fixed and that of Ca2+ is varied, or the other way around. Inspired by the quantitative approach developed by Dagher et al. , Brohus et al. designed a fluorescence anisotropy (FA)-based titration method that permitted a fast exploration of the Ca2+-dependency of the interaction between CaM and four putative RyR2 binding domains, while varying [Ca2+] over a broad range of values, including the physiological levels in cardiomyocytes.
Peptides mimicking the four putative CaM-binding domains in RyR2 were synthesized with a fluorescent N-terminal 5-carboxytetramethylrhodamine (5-TAMRA) label and used in titration experiments with CaM, or its individual N- and C-domains, at 16 discrete values of [Ca2+] . The assay detects the change in the FA signal of the 5-TAMRA-labelled peptide occurring upon CaM (or CaM domain) binding. Since a 16 × 24 wells plate is used (Figure 2), a thorough range of concentration of CaM can be tested (23 serial dilution steps across the columns of one plate row) while keeping the concentration of the interacting peptide constant (∼50 nM) and varying the [Ca2+] discretely in the 16 rows, in the 0.3 nM–400 μM range. Overall, each microtiter plate allows for 24 titration points for CaM concentration at each of the 16 different values of [Ca2+], thus providing 16 CaM-peptide titration curves (Figure 2) in less than 50 min. For most peptides used to mimic the four CaM-binding domains in RyR2 the binding data fit well a simple 1 : 1 interaction model, and the plot of the FA signal as a function of protein concentration at each [Ca2+] (row) showed a sigmoidal trend representing the fractional saturation of the peptide with CaM, finally yielding an apparent KD value at a specific [Ca2+]. By fitting each row-wise titration curve, KD as a function of [Ca2+] could be obtained by nonlinear regression (Figure 2). The authors could also estimate the apparent affinity for Ca2+ of the CaM-peptide complexes by monitoring FA signals as a function of [Ca2+] (column-wise curves) at specific CaM : peptide ratios, ensuring an excess of CaM. By performing a curve fitting to an empirical Hill model, they could estimate the apparent KD of Ca2+-binding to the CaM-peptide complex and the cooperativity of the process described by the Hill coefficient .
The well-designed experiments reported by Brohus et al.  permit a wide set of different conditions to be monitored simultaneously, and provide a direct methodology to test which domain of CaM preferentially binds to a specific RyR2 region over a physiological range of [Ca2+]. Based on their exhaustive screening, the authors were able to build up a detailed dynamic model of domain-wise interaction of CaM with RyR2, and proposed a specific binding scheme that follows [Ca2+] increases from 3 nM up to >10 μM. They concluded that the C-domain of CaM is constitutively bound to the 3581–3611 region of RyR2 in a Ca2+-saturated state, while the N-terminal domain may serve as a dynamic Ca2+ sensor that could either bridge non-adjacent regions of RyR2 or clamp down onto the same interacting region as the C-domain. In a subsequent recent work , the same authors in collaboration with other groups solved the X-ray and NMR structures of CaM in complex with a peptide covering the same 3581–3611 RyR2 region. The complex showed very similar structural properties when comparing wild-type CaM and the N53I mutant associated with CPVT, which demonstrates a tight binding of the peptide to both variants that involves both the N and C-terminals; moreover, the study suggested an alteration of the interaction of the N-terminal domain of CaM with RyR2 induced by the mutant . Recent cryoEM determinations are substantially in line with these findings . Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains of RyR2 . These high-resolution structures confirmed that the shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM rather than to RyR2, which substantially matches the mechanism proposed by Brohus et al. .
The fine regulation of the heartbeat depends critically on very precise affinity and kinetics of the RyR2-CaM-Ca2+ reciprocal interactions. The work reported by Brohus et al. constitutes a significant methodological and conceptual advance in the virtually simultaneous investigation of this tripartite equilibrium. Besides shedding light on the physiological processes in cardiomyocytes, the study has a more general relevance as, with some modifications, it allows the simultaneous detection of the equilibrium dissociation constant in a protein–protein complex as a function of intercellular [Ca2+]. This has a great potential for the study of Ca2+ signalling in a variety of biochemical pathways, especially considering that CaM is known to interact with more than 300 target proteins.
The author declares that there are no competing interests associated with this manuscript.
Open access for this article was enabled by the participation of University of Verona in an all-inclusive Read & Publish pilot with Portland Press and the Biochemical Society.
This work was supported by a grant from the Italian Ministry for Education, University and Research (PRIN 2017 201744NR8S ‘Integrative tools for defining the molecular basis of the diseases’). I would like to thank Dr. Giuditta Dal Cortivo for critically reading the manuscript.