Electrostatic Interactions Within Loop 1 and the Force Generation Region of Human Cardiac Myosin Affect the Rate of Actomyosin Dissociation and ADP Release

AbstractTwo single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the force-generating region of myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the force-generating region modulate affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. Mutant R694N showed faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus confirming that electrostatic interactions within the force-generating region of human cardiac myosin regulate ADP release and the duration of the strongly bound state of actomyosin.

Download Full-text

Role of Electrostatic Interactions in the Isoform-Specific Rate of ADP Release from Human Cardiac Myosin

Biophysical Journal ◽

10.1016/j.bpj.2017.11.791 ◽

2018 ◽

Vol 114 (3) ◽

pp. 140a-141a

Author(s):

Akhil Gargey ◽

Jinghua Ge ◽

Yaroslav Tkachev ◽

Yuri Nesmelov

Keyword(s):

Electrostatic Interactions ◽

Specific Rate ◽

Cardiac Myosin ◽

Adp Release

Download Full-text

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

10.1101/287425 ◽

2018 ◽

Cited By ~ 2

Author(s):

John A. Rohde ◽

David D. Thomas ◽

Joseph M. Muretta

Keyword(s):

Heart Disease ◽

Hypertrophic Cardiomyopathy ◽

Force Generation ◽

Myosin Isoforms ◽

Cardiac Myosin ◽

Atp Turnover ◽

Important Target ◽

Allosteric Effectors ◽

Adp Release ◽

The U.S

AbstractWe used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not found in the isolated S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single ATP turnover kinetics. A two-headed myosin fragment exhibits distinct auto-inhibited ATP turnover kinetics compared to a single-headed fragment. Mavacamten enhanced this auto-inhibition. It also enhanced auto-inhibition of ADP release. Furthermore, actin changes the structure of the auto-inhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.Significance StatementSmall-molecule allosteric effectors designed to target and modulate striated and smooth myosin isoforms for the treatment of disease show promise in preclinical and clinical trials. Beta-cardiac myosin is an especially important target, as heart disease remains a primary cause of death in the U.S. One prevalent type of heart disease is hypertrophic cardiomyopathy (HCM), which is hypothesized to result from dysregulated force generation by cardiac myosin. Mavacamten is a potent cardiac myosin ATPase activity inhibitor that improves cardiac output in HCM animal models. Our results show that mavacamten selectively stabilizes a two-head dependent, auto-inhibited state of cardiac myosin in solution. The kinetics and energetics of this state are consistent with the auto-inhibited super-relaxed state, previously only observed in intact sarcomeres.

Download Full-text

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation

10.1101/2021.10.08.463735 ◽

2021 ◽

Author(s):

Akhil Gargey Iragavarapu ◽

Yuri Nesmelov

Keyword(s):

Electrostatic Interactions ◽

Myosin Head ◽

Salt Bridge ◽

C2c12 Cells ◽

Atp Binding ◽

Cardiac Myosin ◽

Wild Type ◽

Transient Kinetics ◽

Double Mutation ◽

Adp Release

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven stranded beta sheet within the myosin head. Beta isoform specific salt bridge D208:K450 was previously discovered in the molecular dynamics simulations. It was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulates ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutant of the myosin head construct (843 amino acid residues) were expressed in differentiated C2C12 cells, and the kinetics of ATP induced actomyosin dissociation and ADP release were characterized using transient kinetics spectrophotometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. We observed no effect of loop 1 flexibility on the rate of ADP release from actomyosin.

Download Full-text

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽

10.7554/elife.03680 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 69

Author(s):

Joseph Atherton ◽

Irene Farabella ◽

I-Mei Yu ◽

Steven S Rosenfeld ◽

Anne Houdusse ◽

...

Keyword(s):

Electron Microscopy ◽

Conformational Changes ◽

Catalytic Site ◽

Fundamental Question ◽

Force Generation ◽

Atp Binding ◽

Cellular Functions ◽

Microtubule Binding ◽

Cryo Electron Microscopy ◽

Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

Download Full-text

Electrostatic Interactions within Human Cardiac Myosin Head Modulate Its Kinetics

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2431 ◽

2020 ◽

Vol 118 (3) ◽

pp. 433a

Author(s):

Akhil Gargey ◽

Shiril Bharadwaj ◽

Yaroslav V. Tkachev ◽

Yuri E. Nesmelov

Keyword(s):

Electrostatic Interactions ◽

Myosin Head ◽

Cardiac Myosin

Download Full-text

Altered C10 domain in cardiac myosin binding protein-C results in hypertrophic cardiomyopathy

Cardiovascular Research ◽

10.1093/cvr/cvz111 ◽

2019 ◽

Vol 115 (14) ◽

pp. 1986-1997 ◽

Cited By ~ 5

Author(s):

Diederik W D Kuster ◽

Thomas L Lynch ◽

David Y Barefield ◽

Mayandi Sivaguru ◽

Gina Kuffel ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Protein C ◽

Binding Protein ◽

Force Generation ◽

Contractile Dysfunction ◽

Cardiac Myosin ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Myosin Binding

Abstract Aims A 25-base pair deletion in the cardiac myosin binding protein-C (cMyBP-C) gene (MYBPC3), proposed to skip exon 33, modifies the C10 domain (cMyBP-CΔC10mut) and is associated with hypertrophic cardiomyopathy (HCM) and heart failure, affecting approximately 100 million South Asians. However, the molecular mechanisms underlying the pathogenicity of cMyBP-CΔC10mutin vivo are unknown. We hypothesized that expression of cMyBP-CΔC10mut exerts a poison polypeptide effect leading to improper assembly of cardiac sarcomeres and the development of HCM. Methods and results To determine whether expression of cMyBP-CΔC10mut is sufficient to cause HCM and contractile dysfunction in vivo, we generated transgenic (TG) mice having cardiac-specific protein expression of cMyBP-CΔC10mut at approximately half the level of endogenous cMyBP-C. At 12 weeks of age, significant hypertrophy was observed in TG mice expressing cMyBP-CΔC10mut (heart weight/body weight ratio: 4.43 ± 0.11 mg/g non-transgenic (NTG) vs. 5.34 ± 0.25 mg/g cMyBP-CΔC10mut, P < 0.05). Furthermore, haematoxylin and eosin, Masson’s trichrome staining, as well as second-harmonic generation imaging revealed the presence of significant fibrosis and a greater relative nuclear area in cMyBP-CΔC10mut hearts compared with NTG controls. M-mode echocardiography analysis revealed hypercontractile hearts (EF: 53.4%±2.9% NTG vs. 66.4% ± 4.7% cMyBP-CΔC10mut; P < 0.05) and early diastolic dysfunction (E/E′: 28.7 ± 3.7 NTG vs. 46.3 ± 8.4 cMyBP-CΔC10mut; P < 0.05), indicating the presence of an HCM phenotype. To assess whether these changes manifested at the myofilament level, contractile function of single skinned cardiomyocytes was measured. Preserved maximum force generation and increased Ca2+-sensitivity of force generation were observed in cardiomyocytes from cMyBP-CΔC10mut mice compared with NTG controls (EC50: 3.6 ± 0.02 µM NTG vs. 2.90 ± 0.01 µM cMyBP-CΔC10mut; P < 0.0001). Conclusion Expression of cMyBP-C protein with a modified C10 domain is sufficient to cause contractile dysfunction and HCM in vivo.

Download Full-text