Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Molecular Insights into the MLCK Activation by CaM.

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.

Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

Programming Cell-Derived Vesicles with Enhanced Immunomodulatory Properties.

Tumor-associated macrophages are the predominant immune cells present in the tumor microenvironment and mostly exhibit a pro-tumoral M2-like phenotype. However, macrophage biology is reversible allowing them to acquire an anti-tumoral M1-like phenotype in response to external stimuli. A potential therapeutic strategy for treating cancer may be achieved by modulating macrophages from an M2 to an M1-like phenotype with the tumor microenvironment. Here, programmed nanovesicles are generated as an immunomodulatory therapeutic platform with the capability to re-polarize M2 macrophages toward a proinflammatory phenotype. Programmed nanovesicles are engineered from cellular membranes to have specific immunomodulatory properties including the capability to bidirectionally modulate immune cell polarization. These programmed nanovesicles decorated with specific membrane-bound ligands can be targeted toward specific cell types including immune cells. Macrophage-derived vesicles are engineered to enhance immune cell reprogramming toward a proinflammatory phenotype.

Prediction of Kv11.1 potassium channel PAS-domain variants trafficking via machine learning.

Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) could improve the identification of missense variants in LQTS-linked genes. To do this, we investigated KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or class II (trafficking-deficient) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common phenotype for LQTS-associated variants. Specifically, we used computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and hydrogen bonding pairs, as well as folding free energy scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy (≈75%) which KCNH2 variants do not traffic normally. We conclude that structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to an improvement in classification accuracy. Therefore, this approach should be considered to complement the classification of variant of unknown significance (VUS) in the Kv11.1 channel PASD.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms.

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 calcineurin (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, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM 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.

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling.

The cardiac sarcomere is a cellular structure in the heart that enables muscle cells to contract. Dozens of proteins belong to the cardiac sarcomere, which work in tandem to generate force and adapt to demands on cardiac output. Intriguingly, the majority of these proteins have significant intrinsic disorder that contributes to their functions, yet the biophysics of these intrinsically disordered regions (IDRs) have been characterized in limited detail. In this review, we first enumerate these myofilament-associated proteins with intrinsic disorder (MAPIDs) and recent biophysical studies to characterize their IDRs. We secondly summarize the biophysics governing IDR properties and the state-of-the-art in computational tools toward MAPID identification and characterization of their conformation ensembles. We conclude with an overview of future computational approaches toward broadening the understanding of intrinsic disorder in the cardiac sarcomere.

Calmodulin's Interdomain Linker Is Optimized for Dynamics Signal Transmission and Calcium Binding.

Linkers are ubiquitous in multidomain proteins. These linkers are integral to protein functions, and accumulating evidence suggests that the linkers' versatile roles are encoded in their sequences. However, a molecular picture of how amino acid differences in the linker influence protein function is still lacking. By using extensive Gaussian-accelerated MD coupled with dynamic network analysis, we reveal the molecular bases underlying the linker's role in Calmodulin (CaM), a highly conserved Ca2+-signaling hub in eukaryotes. Three CaM constructs comprising a wild-type linker, a flexible linker (four glycines at position D78-S81), and a rigid linker (four prolines at position D78-S81) were simulated. We show that the flexible linker resembles the wild type in allowing CaM to sample a large ensemble of conformations while the rigid linker confines the sampling. Our simulations recapture experimental observations that target binding enhances the Ca2+ affinity to CaM's EF-hand sites at the N-domain. However, only the wild-type linker can both correctly capture the Ca2+ binding order and maintain the α-helical structure of the domain. The other two constructs either bind Ca2+ in an incorrect order or exhibit unfolding of an N-domain helix. We demonstrate that the wild-type linker achieves these outcomes by transmitting interdomain dynamics efficiently. This was evidenced by stronger (anti)correlations among the linker residues, decoupling of the hydrogen bonds between A1-A15 and V35-E45, and structuring of the N-domain for Ca2+ binding. This decoupling was not evident for the other two constructs. Lastly, we show that the wild-type linker's optimal transmission stems from its thermodynamically favorable strain and solvation relative to the other two constructs. Our results show how the linker sequence tunes CaM function, suggesting possible mechanisms for changes in linker properties such as mutations or post-translational modifications to modulate protein/substrate binding.

Mutation-Specific Differences in Kv7.1 (KCNQ1) and Kv11.1 (KCNH2) Channel Dysfunction and Long QT Syndrome Phenotypes.

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano-Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in KCNQ1 (LQT1) and KCNH2 (LQT2). We move beyond the "benign" to a "pathogenic" binary classification scheme for different KCNQ1 and KCNH2 missense variants and discuss gene- and mutation-specific differences in K+ channel dysfunction, which can predispose people to distinct clinical phenotypes (e.g., concealed, pleiotropic, severe, etc.). We conclude by discussing the emerging computational structural modeling strategies that will distinguish between dysfunctional subtypes of KCNQ1 and KCNH2 variants, with the goal of realizing a layered precision medicine approach focused on individuals.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)2.

Modulation of P2X4 pore closure by magnesium, potassium, and ATP.

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 µs we have investigated how metal-complexed ATP stabilizes the channel's open state and prevents its closing. We have identified two metal-binding sites, Mg2+ and potassium K+, one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP-binding site (MBS2), similar to those characterized in Gulf Coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 impeded the collapse of the open-state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and the pore collapsed. This collapse was apparent when fewer than two ATPs were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study therefore provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+.

Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia: Roles of P2 receptors and CD39 in ATP-stimulated microglia.

Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model's predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP.

Crowding within synaptic junctions influences the degradation of nucleotides by CD39 and CD73 ectonucleotidases.

Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to synaptic junctions degrade nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine. Oftentimes nucleotide degradation occurs in a sequential manner, of which ATP degradation by CD39 and CD73 is a representative example. Here, CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which AMP is dephosphorylated into adenosine by CD73. Hence, the concerted activity of CD39 and CD73 can help shape cellular responses to extracellular ATP. In a previous study, we demonstrated that coupled CD39 and CD73 activity within synapse-like junctions is strongly controlled by the enzymes' co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within synaptic junctions, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39 and CD73 ectonucleotidase activity and, in turn, the availability of intrasynapse ATP. Specifically, we developed a spatially explicit, reaction-diffusion model for the coupled conversion of ATP → AMP and AMP → adenosine in a model synaptic junction with crowders that is solved via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density generally suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder-nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder-nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39 and CD73 ectonucleotidase activity, electrostatics, and crowding within synapses influence the availability of nucleotides for intercellular communication.

Homogenization of Continuum-Scale Transport Properties from Molecular Dynamics Simulations: An Application to Aqueous-Phase Methane Diffusion in Silicate Channels.

Silica-based materials including zeolites are commonly used for wide-ranging applications including separations and catalysis. Substrate transport rates in these materials can significantly influence the efficiency of such applications. Two factors that contribute to transport rates include (1) the porosity of the silicate matrix and (2) nonbonding interactions between the diffusing species and the silicate surface. These contributions generally emerge from disparate length scales, namely, "microscopic" (roughly nanometer-scale) and "macroscopic" (roughly micron-scale), respectively. Here, we develop a simulation framework to estimate the simultaneous impact of these factors on methane mass transport in silicate channels. Specifically, we develop a model of methane transport using homogenization theory to obtain transport parameters valid at length scales of hundreds to thousands of nanometers. These parameters implicitly reflect interactions taking place at fractions of a nanometer. The inputs to the homogenization analysis are data from extensive molecular dynamics simulations that incorporate atomistic-scale interactions, processed to yield local diffusion coefficients and mean force potentials. With this model, we demonstrate how nuances in silicate hydration and silica/methane interactions impact methane diffusion rates in silicate materials, including the effects of silicate surface chemistry such as the presence of silanol groups. The molecular dynamics simulations indicate that methane diffusivity at the silica surface is lower than the bulk-like rates observed at the center of channels of sufficient width. However, potentials of mean force generally evidence attractive methane/silica interactions that enhance diffusion overall. By simultaneously accounting for both of these effects, we show that the effective diffusion coefficient for the nanoporous silicate can be approximately double the value of estimates assuming fully bulk-like behavior in the channel. This study therefore demonstrates the importance of determining diffusion coefficients and potentials of mean force at an atomistic level when estimating transport properties in bulk materials. Importantly, we provide a simple homogenization framework to incorporate these molecular-scale attributes into bulk material transport estimates. This hybrid homogenization/molecular dynamics approach will be of general use for describing small-molecule transport in materials with detailed molecular interactions.

Non-Canonical Interaction between Calmodulin and Calcineurin Contributes to the Differential Regulation of Plant-Derived Calmodulins on Calcineurin.

Calmodulin (CaM) serves as an important Ca2+ signaling hub that regulates many protein signaling pathways. Recently, it was demonstrated that plant CaM homologues can regulate mammalian targets, often in a manner that opposes the impact of the mammalian CaM (mCaM). However, the molecular basis of how CaM homologue mutations differentially impact target activation is unclear. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isoforms, sCaM-1 and sCaM-4, share >90 and ∼78% identity with the mCaM, respectively, and activate CaN with comparable or reduced activity relative to mCaM. We used molecular dynamics (MD) simulations and fluorometric assays of CaN-dependent dephosphorylation of MUF-P to probe whether calcium and protein-protein binding interactions are altered by plant CaMs relative to mCaM as a basis for differential CaN regulation. In the presence of CaN, we found that the two sCaMs' Ca2+ binding properties, such as their predicted coordination of Ca2+ and experimentally measured EC50 [Ca2+] values are comparable to mCaM. Furthermore, the binding of CaM to the CaM binding region (CaMBR) in CaN is comparable among the three CaMs, as evidenced by MD-predicted binding energies and experimentally measured EC50 [CaM] values. However, mCaM and sCaM-1 exhibited binding with a secondary region of CaN's regulatory domain that is weakened for sCaM-4. We speculate that this secondary interaction affects the turnover rate (kcat) of CaN based on our modeling of enzyme activity, which is consistent with our experimental data. Together, our data describe how plant-derived CaM variants alter CaN activity through enlisting interactions other than those directly influencing Ca2+ binding and canonical CaMBR binding, which may additionally play a role in the differential regulation of other mammalian targets.

Anomalous structural dynamics of minimally frustrated residues in cardiac troponin C triggers hypertrophic cardiomyopathy.

Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca2+-regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix (TNNC1-p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca2+ binding. We demonstrate cTnC's αD-helix normally functions as a central hub that controls minimally frustrated interactions, maintaining evolutionarily conserved rigidity of the N-domain. αD-helix perturbation remotely alters conformational dynamics of the N-domain, compromising its structural rigidity. Transgenic mice carrying this cTnC mutation exhibit altered dynamics of sarcomere function and hypertrophic cardiomyopathy. Together, our data suggest that disruption of evolutionary conserved molecular frustration networks by a myofilament protein mutation may ultimately compromise contractile performance and trigger hypertrophic cardiomyopathy.

Assessing the Role of Calmodulin's Linker Flexibility in Target Binding.

Calmodulin (CaM) is a highly-expressed Ca2+ binding protein known to bind hundreds of protein targets. Its binding selectivity to many of these targets is partially attributed to the protein's flexible alpha helical linker that connects its N- and C-domains. It is not well established how its linker mediates CaM's binding to regulatory targets yet. Insights into this would be invaluable to understanding its regulation of diverse cellular signaling pathways. Therefore, we utilized Martini coarse-grained (CG) molecular dynamics simulations to probe CaM/target assembly for a model system: CaM binding to the calcineurin (CaN) regulatory domain. The simulations were conducted assuming a 'wild-type' calmodulin with normal flexibility of its linker, as well as a labile, highly-flexible linker variant to emulate structural changes that could be induced, for instance, by post-translational modifications. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 µs of simulation time; of these, 1.7% sampled the fully-bound state observed in the experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be ka= 8.7 × 108 M-1 s-1, which is similar to the diffusion-limited, experimentally-determined rate of 2.2 × 108 M-1 s-1. Furthermore, our simulations recapitulated its well-known inverse relationship between the association rate and the solution ionic strength. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states which are controlled by the linker flexibility. Therefore, our simulations suggest that variations in the CaM linker's propensity for alpha helical secondary structure can modulate the kinetics of target binding.

Structural Changes beyond the EF-Hand Contribute to Apparent Calcium Binding Affinities: Insights from Parvalbumins.

Members of the parvalbumin (PV) family of calcium (Ca2+) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca2+ affinities and selectivities against competing ions like Mg2+ can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca2+ affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca2+ affinity (≈-18 kcal/mol) relative to the wild type (WT) (≈-22 kcal/mol) and an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol). Additionally, we also examined the binding of Mg2+ to these isoforms, which is much weaker than Ca2+. We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition. While the MSA scores differentiated Mg2+ from Ca2+, they did not indicate that Ca2+ binding affinities at the binding loop differed between the PV isoforms. Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg2+ binding was favored for the S55D/E59D αPV and D94S/G98E αPV variants by -18.32 and -1.65 kcal/mol, respectively. These energies implicate significant contributions to ion binding beyond oxygen coordination at the binding loop, which stemmed from changes in α-helicity, ß-sheet character, and hydrogen bonding. Hence, Ca2+ affinity and selectivity against Mg2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions elsewhere. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by members of the broad family of CBPs.

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients.

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.