High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

Risi, Cristina; Schäfer, Luisa U.; Belknap, Betty; Pepper, Ian; White, Howard D.; Schröder, Gunnar F.; Galkin, Vitold E.

doi:10.1016/j.str.2020.09.013

Cited by 52 publications

(88 citation statements)

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“…As rigor myosin-15 potently stimulates F-actin polymerization (Moreland, 2021), we next compared two longitudinally adjacent actin subunits from our F-actin alone and rigor wild-type actomyosin-15 structures, hypothesizing myosin-15 could regulate actin polymerization by modulating F-actin conformation. Consistent with previously reported actomyosin structures (Gurel et al, 2017; Mentes et al, 2018; Risi et al, 2021; von der Ecken et al, 2016), the overall conformation of actin in the presence and absence of myosin-15 is highly similar, with a global RMSD of 0.51 Å (Figure 4A). We primarily observe remodeling adjacent to the myosin-15 HLH, which is propagated through the structure into a global compression of the filament along the longitudinal axis clearly visible in a morph between the density maps of bare F-actin and rigor actomyosin-15 (Video S1).…”

Section: Resultssupporting

confidence: 90%

“…As has been reported in other cryo-EM studies of actomyosin Mentes et al, 2018;Risi et al, 2021;von der Ecken et al, 2016), the local resolution of these reconstructions is highest in the center of the filament and decreases radially outwards towards the distal tip of the myosin lever arm (Figure S1B). For all three complexes, the resolution of F-actin and the actin-myosin interface extends beyond 3 Å, yielding models with unambiguous side-chain density within these areas (Figures 1D,E; S1B).…”

Section: Cryo-em Reconstructions Of Actomyosin-15 Complexes and Bare F-actinsupporting

confidence: 83%

“…We additionally determined the structure of the jordan deafness mutant bound to F-actin at 3.76 Å resolution (Figures 1B, S1A,B). Our rigor wildtype structure represents a substantial improvement in resolution over previously reported actomyosin reconstructions Mentes et al, 2018;Risi et al, 2021;von der Ecken et al, 2016), facilitating building an accurate atomic model of the myosin-15 motor domain (Figure 1C), which has not, to our knowledge, previously been structurally visualized in the absence or presence of F-actin.…”

Section: Cryo-em Reconstructions Of Actomyosin-15 Complexes and Bare F-actinmentioning

confidence: 78%

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Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

Gong

Jiang

Moreland

et al. 2021

Preprint

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SummaryThe motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament (“F-actin”) assembly and identify a progressive hearing loss mutation (p.D1647G, “jordan”) which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin’s D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. ADP-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin’s structural plasticity in a myosin nucleotide-state dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

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Section: Resultssupporting

confidence: 90%

Section: Cryo-em Reconstructions Of Actomyosin-15 Complexes and Bare F-actinsupporting

confidence: 83%

Section: Cryo-em Reconstructions Of Actomyosin-15 Complexes and Bare F-actinmentioning

confidence: 78%

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Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

Gong

Jiang

Moreland

et al. 2021

Preprint

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“…Instead, electron cryo microscopy (cryo-EM) has proven to be an optimal tool to study filamentous proteins (Pospich & Raunser, 2018) such as the actomyosin complex (Behrmann et al, 2012; von der Ecken, Heissler, Pathan-Chhatbar, Manstein, & Raunser, 2016). To date, the structure of the actomyosin rigor complex has been determined for a variety of myosins (Banerjee et al, 2017; Behrmann et al, 2012; Doran et al, 2020; Fujii & Namba, 2017; Gong et al, 2021; Gurel et al, 2017; Mentes et al, 2018; Risi et al, 2020; Robert-Paganin et al, 2021; Vahokoski et al, 2020; von der Ecken et al, 2016). States other than the nucleotide-free rigor state have proven more difficult to study, mainly due to lower binding affinities and short lifetimes.…”

Section: Introductionmentioning

confidence: 99%

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

Pospich

Sweeney

Houdusse

et al. 2021

Preprint

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The molecular motor myosin undergoes a series of major structural transitions during its force-producing motor cycle. The underlying mechanism and its coupling to ATP hydrolysis and actin binding is only partially understood, mostly due to sparse structural data on actin-bound states of myosin. Here, we report 26 high-resolution cryo-EM structures of the actomyosin-V complex in the strong-ADP, rigor, and a previously unseen post-rigor transition state that binds the ATP analog AppNHp. The structures reveal a high flexibility of myosin in each state and provide valuable insights into the structural transitions of myosin-V upon ADP release and binding of AppNHp, as well as the actomyosin interface. In addition, they show how myosin is able to specifically alter the structure of F-actin. The unprecedented number of high-resolution structures of a single myosin finally enabled us to assemble a nearly complete structural model of the myosin-V motor cycle and describe the molecular principles of force production.

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“…We chose the actomyosin complex as a test system because it was among the first assemblies to be studied by time-resolved cryo-EM (Walker et al, 1995(Walker et al, , 1999. Its kinetics are well understood from biochemical experiments, and equilibrium structures have been determined for a variety of different actomyosin complexes (von der Ecken et al, 2016;Fujii & Namba, 2017;Risi et al, 2021). As part of its catalytic cycle, the myosin motor alternates between states of high and low affinity for filamentous actin (F-actin).…”

Section: Introductionmentioning

confidence: 99%

On-grid and in-flow mixing for time-resolved cryo-EM

Klebl¹,

White²,

Sobott³

et al. 2021

Acta Crystallogr D Struct Biol

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Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5–10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7–700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air–water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.

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High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

Abstract: Highlights d The structure of cardiac actomyosin complex is resolved at near-atomic 3.8 Å resolution d Resultant pseudo-atomic model provides molecular details of actomyosin interface d The structure reveals how mutations in actin and myosin may lead to cardiac diseases

Cited by 52 publications

References 100 publications

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

High-resolution structures of the actomyosin-V complex in three nucleotide states provide insights into the force generation mechanism

On-grid and in-flow mixing for time-resolved cryo-EM

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