Nanosurfer assay dissects β-cardiac myosin and cardiac myosin-binding protein C interactions

Journal of General Physiology

Rassici

et al. 2023

Self Cite

Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin–myosin–tropomyosin filament complex at better than 3.4 Å-resolution in the presence and in the absence of Mg2+·ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the nucleotide-binding site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin, and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin

Journal of General Physiology

Rassici

et al. 2023

Self Cite

“…Porcine cardiac F-actin, with 100% sequence identity to human cardiac α-actin, and human cardiac α-tropomyosin (Tpm1.1), containing an N-terminal Ala-Ser extension, were prepared as previously described ( Orzechowski et al, 2014b ). Wild-type recombinant β-cardiac myosin S1 (residues 1–842) was expressed in C2C12 mammalian cells as previously reported ( Touma et al, 2022 ) following high titer infection of cells by recombinant adenovirus (Vector Biolabs). The construct contained standard C-terminal Avi and FLAG tags for FLAG affinity chromatography purification in order to maximize our yield and purity for cryo-EM studies ( Swenson et al, 2017 ; Tang et al, 2019 ; Tang et al, 2021 ; Touma et al, 2022 ).…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type recombinant β-cardiac myosin S1 (residues 1–842) was expressed in C2C12 mammalian cells as previously reported ( Touma et al, 2022 ) following high titer infection of cells by recombinant adenovirus (Vector Biolabs). The construct contained standard C-terminal Avi and FLAG tags for FLAG affinity chromatography purification in order to maximize our yield and purity for cryo-EM studies ( Swenson et al, 2017 ; Tang et al, 2019 ; Tang et al, 2021 ; Touma et al, 2022 ). Mutant human β-cardiac myosin S1 in which amino acids 366–372 were replaced by glycine residues was expressed by the same method.…”

Section: Methodsmentioning

confidence: 99%

Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation

Journal of General Physiology

Pavadai

et al. 2022

Self Cite

During force-generating steps of the muscle crossbridge cycle, the tip of the myosin motor, specifically loop-4, contacts the tropomyosin cable of actin filaments. In the current study, we determined the corresponding effect of myosin loop-4 on the regulatory positioning of tropomyosin on actin. To accomplish this, we compared high-resolution cryo-EM structures of myosin S1-decorated thin filaments containing either wild-type or a loop-4 mutant construct, where the seven-residue portion of myosin loop-4 that contacts tropomyosin was replaced by glycine residues, thus removing polar side chains from residues 366–372. Cryo-EM analysis of fully decorated actin-tropomyosin filaments with wild-type and mutant S1, yielded 3.4–3.6 Å resolution reconstructions, with even higher definition at the actin-myosin interface. Loop-4 densities both in wild-type and mutant S1 were clearly identified, and side chains were resolved in the wild-type structure. Aside from loop-4, actin and myosin structural domains were indistinguishable from each other when filaments were decorated with either mutant or wild-type S1. In marked contrast, the position of tropomyosin on actin in the two reconstructions differed by 3 to 4 Å. In maps of filaments containing the mutant, tropomyosin was located closer to the myosin-head and thus moved in the direction of the C-state conformation adopted by myosin-free thin filaments. Complementary interaction energy measurements showed that tropomyosin in the mutant thin filaments sits on actin in a local energy minimum, whereas tropomyosin is positioned by wild-type S1 in an energetically unfavorable location. We propose that the high potential energy associated with tropomyosin positioning in wild-type filaments favors an effective transition to B- and C-states following release of myosin from the thin filaments during relaxation.

“…Recombinant human β-cardiac myosin S1, including residues 1-842 and containing C-terminal Avi and FLAG tags, was expressed in C2C12 mammalian cells and purified with FLAG affinity chromatography(Tang et al, 2019; Swenson et al, 2017). The initial recombinant adenovirus was purchased from Vector Biolabs (Malvern, PA) and amplified to generate high-titer stocks for infection of C2C12 cells (Touma et al, 2022; Liu et al, 2015). The purified myosin contained mouse skeletal muscle regulatory and essential light chains(Swenson et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin

Rasicci

et al. 2022

Preprint

Following binding to the thin filament, β-cardiac myosin couples ATP-hydrolysis to conformational rearrangements in the myosin motor that drive myofilament sliding and cardiac ventricular contraction. However, key features of the cardiac-specific actin-myosin interaction remain uncertain, including the structural effect of ADP release from myosin, which is rate-limiting during force generation. In fact, ADP release slows under experimental load or in the intact heart due to the afterload, thereby adjusting cardiac muscle power output to meet physiological demands. To further elucidate the structural basis of this fundamental process, we used a combination of cryo-EM reconstruction methodologies to determine structures of the human cardiac actin-myosin-tropomyosin filament complex at better than 3.4 Å resolution in the presence and in the absence of Mg2+-ADP. Focused refinements of the myosin motor head and its essential light chains in these reconstructions reveal that small changes in the active site are coupled to significant rigid body movements of the myosin converter domain and a 16-degree lever arm swing. Our structures provide a mechanistic framework to understand the effect of ADP binding and release on human cardiac β-myosin and offer insights into the force-sensing mechanism displayed by the cardiac myosin motor.