Biology subjects

James A Spudich

Publications and source records attributed to James A Spudich.

3 recordsLinked to original sources

bioRxiv2016

Mutations in the catalytic domain of human β-cardiac myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in {beta}-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human {beta}-cardiac myosin at the molecular level. We observe extremely large increases (25% - 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant myosin motors compared to wild type myosin. In contrast to previous studies of HCM-causing mutations in human {beta}-cardiac myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human {beta}-cardiac myosin.

Biophysics↗

bioRxiv2016

Beyond the myosin mesa: a potential unifying hypothesis on the underlying molecular basis of hyper-contractility caused by a majority of hypertrophic cardiomyopathy mutations

AbstractHypertrophic cardiomyopathy (HCM), the most commonly occurring inherited cardiovascular disease, is primarily caused by mutations in human {beta}-cardiac myosin and myosin binding protein-C. It has been thought that such mutations in myosin increase the intrinsic force of the motor, its velocity of contraction, or its ATPase activity, giving rise to hyper-contractility. We hypothesize that while these parameters are mildly affected by most myosin HCM-causing mutations, a major effect of a majority of myosin HCM mutations is likely to involve an increase in the number of myosin heads that are functionally accessible (Na) for interaction with actin in the sarcomere. We consider a model involving three types of interactions involving the myosin mesa and the converter domain of the myosin motor that hold myosin heads in a sequestered state, likely to be released in a graded manner as the demands on the heart increase: 1) the two myosin heads binding to one another, 2) one head binding to its own coiled-coil tail, and 3) the other head binding to myosin binding protein-C. In addition there is clear evidence of interaction between the coiled-coil tail of myosin and myosin binding protein-C. Experimentally, here we focus on myosin head binding to its own coiled-coil tail and to myosin binding protein-C. We show that phosphorylation of the myosin regulatory light chain and myosin binding protein-C weaken these respective associations, consistent with known enhancements of sarcomere function by these phosphorylations. We show that these interactions are weakened as a result of myosin HCM mutations, in a manner consistent with our structural model. Our data suggests a potential unifying hypothesis for the molecular basis of hyper-contractility caused by human hypertrophic cardiomyopathy myosin mutations, whereby the mutations give rise to an increase in the number of myosin heads that are functionally accessible for interaction with actin in the sarcomere, causing the hyper-contractility observed clinically.

Biochemistry↗

bioRxiv2016

Multi-dimensional structure function relationships in human β-cardiac myosin from population scale genetic variation.

Myosin motors are the fundamental force-generating element of muscle contraction. Variation in the human {beta}-cardiac myosin gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 HCM patients to elucidate novel structure-function relationships within {beta}-cardiac myosin. We first developed computational models of the human {beta}-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein three-dimensional space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified another region enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as a myosin mesa. This surface is prominent in the pre-stroke model, but substantially reduced in size following the power stroke. Notably, HCM patients with variants in the enriched regions have earlier presentation and worse outcome than those with variants in other regions. In summary, this study provides a model for the combination of protein structure, large-scale genetic sequencing and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.

Genomics↗