Ruth F. Sommese

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

Force parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of molecular motor function. Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.

Effects of troponin T cardiomyopathy mutations on the calcium sensitivity of the regulated thin filament and the actomyosin cross-bridge kinetics of human β-cardiac myosin.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca2+-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca2+ sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca2+ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.

Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

Mechanistic Heterogeneity in Contractile Properties of α-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies

Background: Single residue substitutions in sarcomeric proteins cause most inherited cardiomyopathies. Results: Mutant α-tropomyosins cause multiple functional alterations in actin affinity and Ca2+ sensitivity. Conclusion: Mutants follow distinct mechanisms to change Ca2+ sensitivity. Significance: Fluorescence assays to measure changes in troponin C conformation may provide a simple platform for preliminary high throughput screening of modulatory small molecules to treat inherited cardiomyopathies. The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca2+ sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca2+ activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca2+ concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

ABSTRACT Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. Summary: The underlying molecular basis of genetic-based cardiomyopathy diseases is largely unknown. This review describes recent molecular studies that have used human cardiac proteins to begin to elucidate the mechanisms whereby mutations cause disease.

Helicity of short E-R/K peptides

Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide‐based drug design. Previous studies of synthetic Ala‐based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E‐R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E‐R/K–based peptides show significant helix content. Peptides containing one or more E‐R interactions display greater helicity than those with similar E‐K interactions. Significant helicity is achieved in Arg‐based E‐R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E‐R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.

Tuning myosin-driven sorting on cellular actin networks

Myosin V and VI are antagonistic motors that cohabit membrane vesicles in cells. A systematic study of their collective function, however, is lacking and forms the focus of this study. We functionally reconstitute a two-dimensional actin-myosin interface using myosin V and VI precisely patterned on DNA nanostructures, in combination with a model keratocyte actin meshwork. While scaffolds display solely unidirectional movement, their directional flux is modulated by both actin architecture and the structural properties of the myosin lever arm. This directional flux can be finely-tuned by the relative number of myosin V and VI motors on each scaffold. Pairing computation with experimental observations suggests that the ratio of motor stall forces is a key determinant of the observed competitive outcomes. Overall, our study demonstrates an elegant mechanism for sorting of membrane cargo using equally matched antagonistic motors, simply by modulating the relative number of engagement sites for each motor type. DOI: http://dx.doi.org/10.7554/eLife.05472.001

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient. Instead, each regulatory domain (C1a, C1b, and C2) appears to strengthen the pseudosubstrate-catalytic domain interaction in a nucleotide-dependent manner. The pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. Modulating this interaction between the catalytic and regulatory domains reveals a direct correlation between autoinhibition and membrane translocation following PKC activation.

Priming GPCR signaling through the synergistic effect of two G proteins

Significance In this study, we uncover a G-protein–coupled receptor (GPCR) priming mechanism that results from the synergistic effects of two distinct G proteins. Although recent structural and spectroscopic studies of GPCR structure reveal a broad receptor conformational landscape, G-protein activation and downstream signaling are still viewed through the lens of individual ternary complexes between ligand, receptor, and individual effectors. Instead, our findings suggest positive interference between otherwise-disparate signaling pathways that can impact both the potency of GPCR ligands and their cell type-specific responses. Although individual G-protein–coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR–G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand–GPCR–G-protein complex. Here, we present evidence that individual GPCR–G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term “GPCR priming.” Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.