Electrostatic Interactions within Human Cardiac Myosin Head Modulate Its Kinetics

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.

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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.

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Time-Resolved FRET Confirms Human Cardiac Myosin Head-Tail Interaction

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2402 ◽

2020 ◽

Vol 118 (3) ◽

pp. 427a

Author(s):

Alexandra N. Hurst ◽

Shiril Bhardwaj ◽

Akhil Gargey ◽

Yuri Nesmelov

Keyword(s):

Myosin Head ◽

Cardiac Myosin ◽

Time Resolved

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Optical Trapping Shows a 3-Fold Increase in Myosin Head Stiffness by the FHC-Mutation R723G in the β-Cardiac Myosin Heavy Chain

Biophysical Journal ◽

10.1016/j.bpj.2013.11.898 ◽

2014 ◽

Vol 106 (2) ◽

pp. 156a-157a

Author(s):

Christoph Werkman ◽

Nils Hahn ◽

Antonio Francino ◽

Francesc Navarro-Lopéz ◽

Theresia Kraft ◽

...

Keyword(s):

Myosin Heavy Chain ◽

Heavy Chain ◽

Optical Trapping ◽

Myosin Head ◽

Fold Increase ◽

Cardiac Myosin

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Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

10.1101/537159 ◽

2019 ◽

Cited By ~ 4

Author(s):

Arjun S. Adhikari ◽

Darshan V. Trivedi ◽

Saswata S. Sarkar ◽

Dan Song ◽

Kristina B. Kooiker ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Functional Data ◽

Catalytic Domain ◽

Myosin Head ◽

Intramolecular Interactions ◽

Cardiac Myosin ◽

Myosin S1 ◽

Force Velocity ◽

Cardiomyopathy Mutations ◽

Contractility Of The Heart

AbstractHypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

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Electrostatic interactions in the force-generating region of the human cardiac myosin modulate ADP dissociation from actomyosin

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2019.01.045 ◽

2019 ◽

Vol 509 (4) ◽

pp. 978-982 ◽

Cited By ~ 1

Author(s):

Akhil Gargey ◽

Jinghua Ge ◽

Yaroslav V. Tkachev ◽

Yuri E. Nesmelov

Keyword(s):

Electrostatic Interactions ◽

Cardiac Myosin

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Study of Hcm Causing β-Cardiac Myosin Mutations Located at Different Structurally Significant Regions of the Myosin-Head

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2441 ◽

2020 ◽

Vol 118 (3) ◽

pp. 435a

Author(s):

Debanjan Bhowmik ◽

Neha Nandwani ◽

Kathleen Ruppel ◽

Chao Liu ◽

James A. Spudich

Keyword(s):

Myosin Head ◽

Cardiac Myosin

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2P140 Contribution of hydrophobia and electrostatic interactions in the biased motion of myosin head along actin filament(Molecular motors,Poster Presentations)

Seibutsu Butsuri ◽

10.2142/biophys.47.s148_1 ◽

2007 ◽

Vol 47 (supplement) ◽

pp. S148

Author(s):

Tomoki P. Terada ◽

Masaki Sasai

Keyword(s):

Actin Filament ◽

Molecular Motors ◽

Electrostatic Interactions ◽

Myosin Head ◽

Poster Presentations

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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

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Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes

eLife ◽

10.7554/elife.24634 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 61

Author(s):

Lorenzo Alamo ◽

James S Ware ◽

Antonio Pinto ◽

Richard E Gillilan ◽

Jonathan G Seidman ◽

...

Keyword(s):

Energy Consumption ◽

Rare Variants ◽

Myosin Head ◽

Cardiac Myosin ◽

Electrostatic Charges ◽

Protein Partners ◽

Diastolic Relaxation ◽

Interacting Heads Motif ◽

Sarcomere Proteins ◽

Contraction And Relaxation

Cardiac β-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures predict contractility effects but not the prominent relaxation and energetic deficits that characterize HCM. During relaxation, pairs of myosins form interacting-heads motif (IHM) structures that with other sarcomere proteins establish an energy-saving, super-relaxed (SRX) state. Using a human β-cardiac myosin IHM quasi-atomic model, we defined interactions sites between adjacent myosin heads and associated protein partners, and then analyzed rare variants from 6112 HCM and 1315 DCM patients and 33,370 ExAC controls. HCM variants, 72% that changed electrostatic charges, disproportionately altered IHM interaction residues (expected 23%; HCM 54%, p=2.6×10−19; DCM 26%, p=0.66; controls 20%, p=0.23). HCM variant locations predict impaired IHM formation and stability, and attenuation of the SRX state - accounting for altered contractility, reduced diastolic relaxation, and increased energy consumption, that fully characterizes HCM pathogenesis.

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