Biology subjects

Greenberg, M. J.

Publications and source records attributed to Greenberg, M. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Molecular and Cellular Determinants of Human Iron Overload Cardiomyopathy

Iron overload cardiomyopathy (IOC) is a serious heart condition that is caused by elevated levels of systemic iron. IOC is characterized by both systolic and diastolic dysfunction as well as arrhythmias. It has been challenging to isolate the cardiac-specific cellular and molecular mechanisms driving IOC because the disease affects multiple interconnected organ systems. Here, we leverage stem cell technologies, cardiac tissue engineering, and protein reconstitution assays to model key aspects of human IOC in vitro and to probe the cellular and molecular mechanisms driving cardiac dysfunction. We demonstrate that human engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced systolic function, impaired diastolic function, and increased prevalence of arrhythmogenic events. We demonstrate that while both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, leading to reduced viability, cardiomyocytes show higher levels of iron accumulation and higher levels of reactive oxygen species production. Moreover, we show that in a tissue, iron overload has little effect on the action potential kinetics; however, it directly impacts the amplitude and kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, we demonstrate that iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and direct iron-based inhibition of myosin. In summary, our results reveal new insights into the cellular and molecular mechanisms of human IOC pathogenesis, and they establish new in vitro models that can be harnessed to faithfully recapitulate key aspects of the human disease phenotype. HighlightsO_LIContractile aspects of iron overload cardiomyopathy have been difficult to study in vitro. C_LIO_LIWe developed engineered heart tissues to model key aspects of the human disease. C_LIO_LIIn vitro iron overload reduces contractility and induces arrhythmogenesis. C_LIO_LIIron differentially affects cardiomyocytes and cardiac fibroblasts. C_LIO_LIIron overload directly impacts the actomyosin contractile apparatus. C_LI

biophysics↗

bioRxiv2024

Danicamtiv reduces myosin's working stroke but enhances contraction by activating the thin filament

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-year transplant-free survival rate is only [~]50-70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. One such molecule, danicamtiv, recently entered phase II clinical trials; however, its mechanism of action and direct effects on myosins mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosins working stroke, and in contrast to studies in muscle fibers, we found that it does not affect actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically-relevant calcium concentrations. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges contribute to increased cardiac contraction and improved diastolic function compared to the related myotrope, omecamtiv mecarbil. Taken together, our results have important implications for the design of new sarcomeric-targeting compounds for heart failure. SIGNIFICANCE STATEMENTHeart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.

biophysics↗

bioRxiv2024

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. The method has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 {micro}g sample input, with 235 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

biochemistry↗

bioRxiv2024

Structural dynamics of the intrinsically disordered linker region of cardiac troponin T

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponins function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Forster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the {Delta}E160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies. SIGNIFICANCE STATEMENTTroponin plays a central role in regulating heart contraction, and mutations in troponin can cause human cardiomyopathies. There are several functionally-significant regions of troponin that have not been structurally resolved, including the troponin T linker region that contains multiple cardiomyopathy mutations. In these unresolved regions, it is not possible to understand how changes in sequence affect function. We used computational and experimental techniques to demonstrate that this linker is dynamic and intrinsically disordered both in isolation and in the fully regulated thin filament. Moreover, we show how a cardiomyopathy mutation in this region affects function via allosteric disruption of intermolecular interactions. Our results highlight the need to consider how key mutations affect troponin disorder rather than the structure-function relationship.

biophysics↗

bioRxiv2024

Harnessing molecular mechanism for precision medicine in dilated cardiomyopathy caused by a mutation in troponin T

Familial dilated cardiomyopathy (DCM) is frequently caused by autosomal dominant point mutations in genes involved in diverse cellular processes, including sarcomeric contraction. While patient studies have defined the genetic landscape of DCM, genetics are not currently used in patient care, and patients receive similar treatments regardless of the underlying mutation. It has been suggested that a precision medicine approach based on the molecular mechanism of the underlying mutation could improve outcomes; however, realizing this approach has been challenging due to difficulties linking genotype and phenotype and then leveraging this information to identify therapeutic approaches. Here, we used multiscale experimental and computational approaches to test whether knowledge of molecular mechanism could be harnessed to connect genotype, phenotype, and drug response for a DCM mutation in troponin T, deletion of K210. Previously, we showed that at the molecular scale, the mutation reduces thin filament activation. Here, we used computational modeling of this molecular defect to predict that the mutant will reduce cellular and tissue contractility, and we validated this prediction in human cardiomyocytes and engineered heart tissues. We then used our knowledge of molecular mechanism to computationally model the effects of a small molecule that can activate the thin filament. We demonstrate experimentally that the modeling correctly predicts that the small molecule can partially rescue systolic dysfunction at the expense of diastolic function. Taken together, our results demonstrate how molecular mechanism can be harnessed to connect genotype and phenotype and inspire strategies to optimize mechanism-based therapeutics for DCM. Significance statementDilated cardiomyopathy (DCM), a leading cause of heart failure, is characterized by the inability of the heart to perfuse the body at normal filling pressures. There are multiple causes of DCM, including point mutations in sarcomeric proteins, but most patients receive similar courses of treatment, regardless of the underlying cause of the DCM. Many patients remain unserved by current therapies, and there is a need for new approaches. Here, we use multiscale experimental and computational approaches to demonstrate how knowledge of molecular mechanism can be harnessed to accurately predict the effects of a patient-specific mutation and responses to presumptive therapeutics. Our approach lays the foundation for a precision medicine approach to DCM.

biophysics↗

bioRxiv2024

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility

Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1s high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only [~]20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 mutation, R256H. We previously identified this mutation in multiple family members with dilated cardiomyopathy (DCM), who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent functional effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using Cryo-EM, and we see alterations in actins structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+ human induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric disorganization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy. Significance StatementSkeletal muscle actin mutations are well-known to cause skeletal myopathies, but their role in cardiomyopathies have been controversial as skeletal muscle actin is only expressed at modest levels in the heart. Here, we demonstrate that a skeletal muscle actin mutation potently causes multiple defects in actin function at the atomic and molecular scales, and it functions in a dominant fashion, leading to cardiomyocyte contractile defects. Our results establish how skeletal muscle actin mutations may cause cardiomyocyte dysfunction and lay the foundation for future studies of the role of skeletal muscle actin in cardiomyopathy.

biophysics↗

bioRxiv2024

Insights into post-translational regulation of skeletal muscle contractile function by the acetyltransferases, p300 and CBP

Mice with skeletal muscle-specific inducible double knockout of the lysine acetyltransferases, p300 (E1A binding protein p300) and CBP (cAMP-response element-binding protein binding protein), referred to as i-mPCKO, demonstrate a dramatic loss of contractile function in skeletal muscle and ultimately die within 7 days. Given that many proteins involved in ATP generation and cross-bridge cycling are acetylated, we investigated whether these processes are dysregulated in skeletal muscle from i-mPCKO mice and thus could underlie the rapid loss of muscle contractile function. Just 4-5 days after inducing knockout of p300 and CBP in skeletal muscle from adult i-mPCKO mice, there was [~]90% reduction in ex vivo contractile function in the extensor digitorum longus (EDL) and a [~]65% reduction in in vivo ankle dorsiflexion torque, as compared to wildtype (WT; i.e. Cre negative) littermates. Despite the profound loss of contractile force in i-mPCKO mice, there were no genotype-driven differences in fatigability during repeated contractions, nor were there genotype differences in mitochondrial specific pathway enrichment of the proteome, intermyofibrillar mitochondrial volume or mitochondrial respiratory function. As it relates to cross-bridge cycling, remarkably, the overt loss of contractile function in i-mPCKO muscle was reversed in permeabilized fibers supplied with exogenous Ca2+ and ATP, with active tension being similar between i-mPCKO and WT mice, regardless of Ca2+ concentration. Actin-myosin motility was also similar in skeletal muscle from i-mPCKO and WT mice. In conclusion, neither mitochondrial abundance/function, nor actomyosin cross-bridge cycling, are the underlying driver of contractile dysfunction in i-mPCKO mice. New & NoteworthyThe mechanism underlying dramatic loss of muscle contractile function with inducible deletion of both p300 and CBP in skeletal muscle remains unknown. Here we find that impairments in mitochondrial function or cross-bridge cycling are not the underlying mechanism of action. Future work will investigate other aspects of excitation-contraction coupling, such as Ca2+ handling and membrane excitability, as contractile function could be rescued by permeabilizing skeletal muscle, which provides exogenous Ca2+ and bypasses membrane depolarization.

cell biology↗

bioRxiv2023

Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain ({beta}-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human {beta}-MHC Subfragment 1 (S1) in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical functions, including unchanged ATPase and enhanced actin velocity but had distinct effects on the basal activity of myosin. HCM-I467V S1 showed no change in basal ATPase activity of myosin while LVNC-I467T reduced the basal ATPase activity by 50%. Molecular dynamics simulations reveal that I467T allosterically disrupts nucleotide binding of myosin, which may contribute to the uncoupled reduced basal activity and enhanced actin velocity observed in this mutation. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

biophysics↗

bioRxiv2023

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosins mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosins primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction. Significance StatementHuman heart contraction is powered by the molecular motor {beta}-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of {beta}-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

biophysics↗

bioRxiv2022

Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatins binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). To test our ability to make blind predictions, we predicted blebbistatins binding affinity for an isoform (Myh7b) whose blebbistatin sensitivity was unknown. Encouragingly, we find good agreement between the predicted and measured IC50 (0.67 {micro}M vs. 0.36 {micro}M). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets. SignificanceDrug development requires the discovery of compounds which specifically target one member of a protein family without triggering side effects that arise from interactions with other related proteins. Myosins are a family of motor proteins that are drug targets for heart diseases, cancer, and parasitic infections. Here, we investigate why the compound blebbistatin specifically inhibits some myosins more potently than others, even though its binding site is closed in all known experimental structures. We find that the blebbistatin binding pocket opens in molecular dynamics simulations of certain myosin motors, and that the probability of opening predicts how potently blebbistatin inhibits a particular motor. Our work suggests that differences in cryptic pocket formation can be exploited to develop specific therapeutics.

biophysics↗

bioRxiv2021

Myofilament Glycation in Diabetes Reduces Contractility by Inhibiting Tropomyosin Movement, is Rescued by cMyBPC Domains

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 M MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MGs functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosins blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosins position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF.

biophysics↗

bioRxiv2020

SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

Epidemiological studies of the COVID-19 pandemic have revealed evidence of cardiac involvement and documented that myocardial injury and myocarditis are predictors of poor outcomes. Nonetheless, little is understood regarding SARS-CoV-2 tropism within the heart and whether cardiac complications result directly from myocardial infection. Here, we develop a human engineered heart tissue model and demonstrate that SARS-CoV-2 selectively infects cardiomyocytes. Viral infection is dependent on expression of angiotensin-I converting enzyme 2 (ACE2) and endosomal cysteine proteases, suggesting an endosomal mechanism of cell entry. After infection with SARS-CoV-2, engineered tissues display typical features of myocarditis, including cardiomyocyte cell death, impaired cardiac contractility, and innate immune cell activation. Consistent with these findings, autopsy tissue obtained from individuals with COVID-19 myocarditis demonstrated cardiomyocyte infection, cell death, and macrophage-predominate immune cell infiltrate. These findings establish human cardiomyocyte tropism for SARS-CoV-2 and provide an experimental platform for interrogating and mitigating cardiac complications of COVID-19.

immunology↗

bioRxiv2020

Computational Tool for Ensemble Averaging of Single-Molecule Data

Molecular motors couple chemical transitions to conformational changes that perform mechanical work in a wide variety of biological processes. Disruption of this coupling can lead to diseases, and therefore there is a need to accurately measure mechanochemical coupling in motors in both health and disease. Optical tweezers, with nanometer spatial and millisecond temporal resolution, have provided valuable insights into these processes. However, fluctuations due to Brownian motion can make it difficult to precisely resolve these conformational changes. One powerful analysis technique that has improved our ability to accurately measure mechanochemical coupling in motor proteins is ensemble averaging of individual trajectories. Here, we present a user-friendly computational tool, Software for Precise Analysis of Single Molecules (SPASM), for generating ensemble averages of single-molecule data. This tool utilizes several conceptual advances, including optimized procedures for identifying single-molecule interactions and the implementation of a change point algorithm, to more precisely resolve molecular transitions. Using both simulated and experimental data, we demonstrate that these advances allow for accurate determination of the mechanics and kinetics of the myosin working stroke with a smaller set of data. Importantly, we provide our open source MATLAB-based program with a graphical user interface that enables others to readily apply these advances to the analysis of their own data. Statement of SignificanceSingle molecule optical trapping experiments have given unprecedented insights into the mechanisms of molecular machines. Analysis of these experiments is often challenging because Brownian motion-induced fluctuations introduce noise that can obscure molecular motions. A powerful technique for analyzing these noisy traces is ensemble averaging of individual binding interactions, which can uncover information about the mechanics and kinetics of molecular motions that are typically obscured by Brownian motion. Here, we provide an open source, easy-to-use computational tool, SPASM, with a graphical user interface for ensemble averaging of single molecule data. This computational tool utilizes several conceptual advances that significantly improve the accuracy and resolution of ensemble averages, enabling the generation of high-resolution averages from a smaller number of binding interactions.

biophysics↗

bioRxiv2020

Mechanical dysfunction induced by a hypertrophic cardiomyopathy mutation is the primary driver of cellular adaptation

Familial hypertrophic cardiomyopathy (HCM), a leading cause of sudden cardiac death, is primarily caused by mutations in sarcomeric proteins. The pathogenesis of HCM is complex, with functional changes that span scales from molecules to tissues. This makes it challenging to deconvolve the biophysical molecular defect that drives the disease pathogenesis from downstream changes in cellular function. Here, we examined a HCM mutation in troponin T, R92Q. We demonstrate that the primary molecular insult driving the disease pathogenesis is mutation-induced alterations in tropomyosin positioning, which causes increased molecular and cellular force generation during calcium-based activation. We demonstrate computationally that these increases in force are direct consequences of the initial molecular insult. This altered cellular contractility causes downstream alterations in gene expression, calcium handling, and electrophysiology. Taken together, our results demonstrate that molecularly driven changes in mechanical tension drive the early disease pathogenesis, leading to activation of adaptive mechanobiological signaling pathways.

biophysics↗

bioRxiv2019

Structural dynamics of isolated myosin motor domains encode differences in their mechanochemical cycles

Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for twelve myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally-measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships. Subject AreasBiochemistry and Chemical Biology, Structural Biology and Molecular Physics

biophysics↗

bioRxiv2019

Disrupted mechanobiology links the molecular and cellular phenotypes in familial dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, {Delta}K210. We determined the molecular mechanism of {Delta}K210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that {Delta}K210 not only reduces contractility, but also causes cellular hypertrophy and impairs cardiomyocytes ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

biophysics↗

bioRxiv2019

Computational tool to study perturbations in muscle regulation and its application to heart disease

Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in the sarcomere and generate pulling forces. This process is regulated by calcium, and it can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations (e.g., {beta}-adrenergic stimulation), and pharmacological interventions. Therefore, it is important to have a methodology to robustly determine the mechanism of these perturbations and statistically evaluate their effects. Here, we present an approach to measure the equilibrium constants that govern muscle activation, estimate uncertainty in these parameters, and statistically test the effects of perturbations. We provide a MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials that make this approach accessible. The hypothesis testing and error estimation approaches described here are broadly applicable, and the provided tools work with other types of data, including cellular measurements. To demonstrate the utility of the approach, we apply it to determine the biophysical mechanism of a mutation that causes familial hypertrophic cardiomyopathy. This approach is generally useful for studying the mechanisms of muscle diseases and therapeutic interventions that target muscle contraction.

biophysics↗

bioRxiv2018

Exposons exploit cooperative changes in solvent exposure to detect cryptic allosteric sites and other functionally-relevant conformational transitions

Proteins are dynamic molecules that undergo conformational changes to a broad spectrum of different excited states. Unfortunately, the small populations of these states make it difficult to determine their structures or functional implications. Computer simulations are an increasingly powerful means to identify and characterize functionally-relevant excited states. However, this advance has uncovered a further challenge: it can be extremely difficult to identify the most salient features of large simulation datasets. We reasoned that many functionally-relevant conformational changes are likely to involve large, cooperative changes to the surfaces that are available to interact with potential binding partners. To examine this hypothesis, we introduce a method that returns a prioritized list of potentially functional conformational changes by segmenting protein structures into clusters of residues that undergo cooperative changes in their solvent exposure, along with the hierarchy of interactions between these groups. We term these groups exposons to distinguish them from other types of clusters that arise in this analysis and others. We demonstrate, using three different model systems, that this method identifies experimentally-validated and functionally-relevant conformational changes, including conformational switches, allosteric coupling, and cryptic pockets. Our results suggest that key functional sites are hubs in the network of exposons. As a further test of the predictive power of this approach, we apply it to discover cryptic allosteric sites in two different {beta}-lactamase enzymes that are widespread sources of antibiotic resistance. Experimental tests confirm our predictions for both systems. Importantly, we provide the first evidence for a cryptic allosteric site in CTX-M-9 {beta}-lactamase. Experimentally testing this prediction did not require any mutations, and revealed that this site exerts the most potent allosteric control over activity of any pockets found in {beta}-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 {beta}-lactamase demonstrates the utility of exposons.

biophysics↗