Calmodulin (CaM) is a highly conserved Ca2+ sensor that regulates hundreds of cellular targets through Ca2+ -dependent conformational dynamics. Despite its central role in Ca2+ signaling and disease, its evolutionary conservation and structural flexibility have suggested that CaM is resistant to rational redesign. Here, using the cardiac Ca2+ release channel Ryanodine receptor 2 (RyR2) as a model system, we tested whether incorporating conformational dynamics into computational protein design enables functional reengineering of CaM. We first applied a static structure-based redesign to increase CaM-RyR2 affinity. Although the resulting variant bound more tightly to both the RyR2 peptide and the intact channel in vitro, it distorted peptide geometry and worsened Ca2+ leak in cardiomyocytes ex vivo. Guided by molecular dynamics simulations, we then developed a dynamic-structure redesign strategy that preserves conformational integrity while strengthening binding. The resulting CaM variant exhibited increased RyR2 affinity and reduced pathological Ca2+ leak in a disease-relevant model. These findings show that improved binding affinity alone is insufficient to enhance physiological regulation and that successful CaM redesign requires preservation of conformational dynamics. More broadly, they demonstrate that integrating conformational dynamics into protein redesign can enable functionally predictive engineering of flexible regulatory protein-protein interactions.

Aims This study aims to resolve the mechanisms underlying Calmodulin (CaM)’s signalling diversity by investigating whether the three CaM genes—Calm1, Calm2, and Calm3—play distinct or redundant roles in cardiac myocytes, focusing on their spatial mRNA localization and interactions with key targets. Methods and results We utilized single-molecule mRNA detection and three-dimensional imaging to map the spatial distribution of Calm1, Calm2, and Calm3 mRNAs within ventricular myocytes. These mRNAs were found to be consistently positioned within specific cellular zones, overlapping with their target mRNAs and forming region-specific transcript conjunctions. This spatial organization aligns with two distinct protein synthesis pathways: centralized synthesis near the nucleus for proteins such as Cx43 and localized synthesis in more peripheral cytosolic areas for proteins like RyR2. Ablation of Calm1 triggered compensatory increases in Calm2 and Calm3; however, this compensation was insufficient to restore normal CaM transcript distribution, leading to disrupted Ca²⁺ handling. In the context of hypertrophic heart failure (HF), the distribution and spatial interactions of CaM transcripts, while potentially adaptive to support myocyte growth, become disrupted, leading to disorganized CaM signalling. Conclusion Our findings reveal that Calm1, Calm2, and Calm3 fulfil distinct, non-redundant roles in cardiac myocytes through their spatially regulated mRNA localization (spatiotemporal coding). This precise spatial control of mRNA localization is critical for region-specific CaM signalling and is disrupted in hypertrophic HF, contributing to pathological remodelling.

Calmodulin, a protein that affords Ca2+-sensitivity to multiple enzymes and ion channels, is encoded by three distinct genes. Recent studies revealed that mutations in only one out of six alleles can underlie cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). Calm CPVT mutations are thought to disrupt RyR2 function, while LQTS mutations were linked to a reduction in L-type Ca2+-channel Ca2+-dependent inactivation. Surprisingly, mutations in different Calm genes resulting in identical protein products can lead to different arrhythmia phenotypes.

Calmodulin (CaM) is a vital calcium-binding protein encoded by three distinct CALM genes, all producing the same protein. Although each gene has its unique regulatory domain, whether they are redundant or play specialized roles is unknown. To gain deeper insights into their functional interplay, we examined the spatiotemporal expression patterns of Calm1,2,3, and selected CaM target genes' mRNAs under the influence of isoproterenol (ISO), a beta-adrenergic receptor agonist, which modulates CaM signaling.

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase cal-cineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, indi-vidual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaN-CaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering systolic calcium concentration, thereby strengthening cardiac function. Here, we examined the effect of our previously identified calcium sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This led to the identification of three novel low affinity binders, which had similar binding affinities to known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM .

Introduction: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a stress-induced arrhythmic syndrome due to genetic defects of the sarcoplasmic reticulum (SR) Ca release channel complex of ryanodine receptor 2 (RyR2). Recent studies suggest that mitochondria function as a protective Ca buffer to absorb RyR2-mediated aberrant Ca release in CPVT. However, the molecular mechanism underlying the protective Ca buffering function of CPVT mitochondria remains unclear. Hypothesis: We hypothesize that the tethering between SR and mitochondria, also known as mitochondria-associated-membranes (MAMs) are promoted in CPVT to facilitate SR-mitochondria Ca transfer. Moreover, manipulating SR-mitochondria tethering or MAMs impacts arrhythmogenesis in CPVT. Methods: Ventricular myocytes were isolated from a CPVT model of CASQ2 knockout (Cnull) mouse. Cellular immunofluorescence assays and western blots were employed to detect MAMs remodeling in CPVT cells. Pharmacological approach was employed to disrupt MAMs and examine its effect on cellular arrhythmogenesis using live-cell imaging. Results: SR-mitochondria tethering was assessed by examining the interaction between RyR2 and Voltage-dependent anion channel (VDAC), a protein localized in the mitochondrial-outer-membrane. As compared with wild-type (WT), we detected a higher degree of colocalization of immunofluorescence between RyR2 and VDAC in Cnull cells, as well as increased RyR2-VDAC interactions by proximity ligation assay. The expression of SR-mitochondria tethering protein Mitofusin2 was increased in MAMs isolated from CPVT hearts. Colchicine was employed to induce a partial disruption of MAMs in Cnull cells. In intact Cnull cells perfused with β agonist isoproterenol, colchicine exacerbated arrhythmogenic Ca waves. In permeabilized cells, colchicine increased the frequency of arrhythmogenic Ca waves and reduced mitochondrial Ca uptake. Conclusions: Our results support that MAMs are promoted in CPVT cells to facilitate SR-mitochondria Ca transfer so mitochondria can function as a protective Ca buffer. Pharmacological disruption of SR-mitochondria tethering limits SR-mitochondria Ca transfer, thus exacerbating cellular arrhythmic burden.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.