Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity

Gurel, Pinar S.; Kim, Laura Y.; Ruijgrok, Paul V.; Omabegho, Tosan; Bryant, Zev; Alushin, Gregory M.

doi:10.7554/elife.31125

Cited by 67 publications

(159 citation statements)

References 118 publications

(231 reference statements)

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“…The ATP-dependence of this enhanced F-actin binding strongly suggests it is activated by force, rather than allosteric remodeling of actin filament structure due to local deformations imposed by motor binding (Gurel et al, 2017). Furthermore, visual inspection supports increased F-actin bundling by both vinculin and α-catenin upon motor activation ( Fig.…”

Section: Physiological Forces Generated By Myosin Motors Activate F-amentioning

confidence: 80%

“…Filamentous actin (F-actin) was polymerized fresh for each experiment from actin monomers in G-Mg (G buffer supplemented with 0.1 mM MgCl2) and KMEI (50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole pH 7.0, 1mM DTT) buffers as described previously (Gurel et al, 2017). 30% rhodaminelabeled actin filaments used in TIRF microscopy assays were prepared by co-polymerizing unlabeled actin monomers and rhodamine-labeled actin monomers (Cytoskeleton catalogue # AR05) at a 7:3 molar ratio (total actin concentration 1µM) at room temperature for 2 hours in the dark.…”

Section: F-actin Preparationmentioning

confidence: 99%

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Molecular mechanism for direct actin force-sensing by α-catenin

Mei

Reyes

Reynolds

et al. 2020

Preprint

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The actin cytoskeleton mediates mechanical coupling between cells and their tissue microenvironments. The architecture and composition of actin networks are modulated by force, but it is unclear how interactions between actin filaments (F-actin) and associated proteins are mechanically regulated. Here, we employ both optical trapping and biochemical reconstitution with myosin motor proteins to show force greater than one piconewton applied solely to F-actin enhances binding by the essential cell-cell adhesion protein αE-catenin, but not its homolog vinculin. Near atomic-resolution cryoelectron microscopy structures of both proteins bound to F-actin reveal unique rearrangements that facilitate their flexible C-termini refolding to engage distinct interfaces. Truncating α-catenin's C-terminus eliminates force-activated F-actin binding, and addition of this motif to vinculin confers force-activated binding, demonstrating that α-catenin's C-terminus is a modular detector of F-actin tension. Our studies establish that piconewton force on F-actin can enhance partner binding, which we propose mechanically regulates cellular adhesion through a-catenin.

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Section: Physiological Forces Generated By Myosin Motors Activate F-amentioning

confidence: 80%

Section: F-actin Preparationmentioning

confidence: 99%

Molecular mechanism for direct actin force-sensing by α-catenin

Mei

Reyes

Reynolds

et al. 2020

Preprint

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“…This is in line with previous studies reporting that myosin-5 141 and myosin-6 can distinguish the ADP-Pi from the ADP state in phalloidin stabilized F-actin 142 (Zimmermann et al, 2015). Considering our previous data indicating that the ABP coronin can 143 sense the nucleotide state of F-actin via the D-loop conformation , we 144 propose that myosin-5 and myosin-6 can read the age of F-actin in a similar manner by binding 145 to the D-loop C-terminus interface (Gurel et al, 2017;S. F. Wulf et al, 2016).…”

Section: Introduction 34mentioning

confidence: 80%

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Pospich

Merino

Raunser

2019

Preprint

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Actin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.

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“…In Figure 1A, the position of ATP is based on ligand-bound crystal structure 1MMA (Gulick et al, 1997). The actin binding region was defined by all atoms within 10 Å of the actin filament after alignment to 6BNP chain K (Gurel et al, 2017).…”

Section: Methodsmentioning

confidence: 99%

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties

Porter

Meller

Zimmerman

et al. 2019

Preprint

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Myosin motor domains perform an extraordinary diversity of biological functions despite sharing a common mechanochemical cycle. Motors are adapted to their function, in part, by tuning the thermodynamics and kinetics of steps in this cycle. However, it remains unclear how sequence encodes these differences, since biochemically distinct motors often have nearly indistinguishable crystal structures. We hypothesized that sequences produce distinct biochemical phenotypes by modulating the relative probabilities of an ensemble of conformations primed for different functional roles. To test this hypothesis, we modeled the distribution of conformations for twelve myosin motor domains by building Markov state models (MSMs) from an unprecedented two milliseconds of all-atom, explicit-solvent molecular dynamics simulations. Comparing motors reveals shifts in the balance between nucleotide-favorable and nucleotide-unfavorable P-loop conformations that predict experimentally-measured duty ratios and ADP release rates better than sequence or individual structures. This result demonstrates the power of an ensemble perspective for interrogating sequence-function relationships.Subject AreasBiochemistry and Chemical Biology, Structural Biology and Molecular Physics

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Cryo-EM structures reveal specialization at the myosin VI-actin interface and a mechanism of force sensitivity

Cited by 67 publications

References 118 publications

Molecular mechanism for direct actin force-sensing by α-catenin

Molecular mechanism for direct actin force-sensing by α-catenin

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Conformational distributions of isolated myosin motor domains encode their mechanochemical properties

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