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

Figures

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  Figure 1.. High-resolution reconstructions of myosin VI bound to actin. (A) Schematic depicting myosin VI states in the force generation cycle. Filament polarity is indicated throughout the paper with pointed end as '-' and barbed end as '+'. Cryo-EM reconstructions of actomyosin VI in the nucleotide-free (rigor) state (left), MgADP (ADP) state (middle), and actin alone (right). Actin, light blue; myosin VI, magenta (rigor) and dark magenta (ADP). (B) Atomistic model of actomyosin VI (rigor) colored corresponding to treatment during HR MDFF. Blue (actin), large side chains and backbone atoms subjected to fitting guided by density map; Green (MD), backbone atoms only subjected to fitting guided by density map; Red (MD loop 2 residues 622–636 and HCM loop residues 397–405), density term disabled due to conformational variability; Grey (ADP and Magnesium ion in actin), fixed atoms. (C) Superposition of the actin nucleotide-binding cleft from all six actomyosin interfaces in the HR MDFF rigor model, docked into the density map and colored by heteroatom. Large side chains and ADP are displayed.

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  Figure 1—figure supplement 1.. Electron microscopy and resolution assessment. (A) Representative micrographs of actin filaments decorated with nucleotide free myosin VI (left), Mg-ADP myosin VI (middle), and in the absence of myosin (right). Scale bar, 100 nm. (B) Fourier Shell Correlation (FSC) curves for reconstructions of actin-MyoVI rigor (left) and ADP (right) with RNA (grey), without RNA (black), and combining the two datasets (magenta). (C) Cartoon ribbon diagram of the engineered myosin VI used in this study. (D) FSC curves for reconstructions of actin-myoVI rigor state (left), ADP state (middle), and bare actin (right). FSC curves approximately corresponding to different domains were generated by radially masking reconstructions at 120 Å (full reconstruction, magenta), 90 Å (truncated MD to exclude lever arm and converter, dark magenta), and 40 Å (actin, light blue). (E) Reconstructions for actin-myoVI rigor (left), actin-myoVI ADP (middle), and bare actin (right) colored according local resolution as determined by ResMap. Radii corresponding to S1D indicated. (F) View of the HR rigor MDFF model (magenta) and the rigor-like state initial model (2BKI, grey) superimposed in the reference frame of the…

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  Figure 2.. Interactions composing the actomyosin VI interface in rigor. (A) All six actomyosin interfaces from the HR MDFF rigor model, superimposed based on the Cα coordinates of the dark-blue actin subunits. MD, magenta; actin subunits, varying shades of blue. (B–D) Detail views of interface contacts suggested by MDFF, colored as in A; EM density map is displayed on left side in transparent grey. (B) Hydrophobic interface between MD HLH and actin SD1/SD3. (C) Milligan contact interactions between MD loop 3 and actin D-loop/SD1. (D) Electrostatic interaction between MD loop 4 and actin SD3. (E) Interface between MD HCM loop and loop 2 with actin surface, colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). (F) Salt bridge formation between the base of the MD HCM loop with actin SD1.

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  Figure 2—figure supplement 1.. Comparison of interactions composing the actomyosin interface between rigor-state myosin IIC and myosin VI. (A) The rigor state myosin IIC actomyosin interface from 5JLH (von der Ecken et al., 2016) consisting of 1 MD (light pink) and three actin subunits (shades of blue), analogous to Figure 2. (B–F) Detailed views of interface contacts, colored as in A; HR rigor MDFF model, panels from Figure 2B–2F, displayed on left side for comparison. (B) Hydrophobic interaction between myosin IIC HLH and actin SD1/SD3. Panel from Figure 2B adjacent for comparison. (C) Milligan contact interactions between myosin IIC loop 3/HLH and actin D-loop/SD1. Panel from Figure 2C adjacent for comparison. (D) Putative electrostatic interactions between myosin IIC loop 4 and actin SD 3. Panel from Figure 2D adjacent for comparison. (E) Interface of myosin IIC HCM loop and loop 2 with actin surface colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). Panel from Figure 2E adjacent for comparison. (F) Interactions at the base of the myosin IIC HCM loop with actin SD1. Panel from Figure 2F adjacent for comparison.

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  Figure 3.. A unique contact is established upon transition from ADP to rigor. (A) View of the LPF APD MDFF model (dark magenta) and LPF rigor MDFF model superimposed in the reference frame of the actin filament (light gray density). To generate this superposition, the ADP and rigor density maps were aligned, then their corresponding atomistic models were rigid body fit into the aligned maps. (B) Minimal actin binding cleft rearrangements are observed between ADP and rigor, superimposed as described in A. ADP U50, magenta; ADP L50, dark magenta; rigor U50, dark grey; rigor L50, light grey; actin density, white. Arrows denote displacements of domain centroids (spheres) from ADP to the rigor state. Centroids were determined for U50 (residues 180–206, 229–397, and 405–441) and L50 (residues 467–597 and 638–661) domains. (C) MDFF indicates the Milligan contact cation-π interaction between R561 in the MD loop 3 and Y91 in the adjacent actin is absent in ADP (left) but is established upon transition to the rigor state (right), with clear density for these sidechains in the rigor map. For both states, all six actomyosin interfaces in the corresponding HR MDFF model are displayed superimposed on one…

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  Figure 3—figure supplement 1.. Comparison of ADP and rigor motor nucleotide binding cleft. (A) View of the MD from the LPF ADP MDFF model (left, dark magenta) and LPF rigor MDFF model (right, light magenta), rigid-body fit into their corresponding segmented density maps (transparent grey) low-pass filtered to 7.5 Å. (B) View of the boxed region indicated in panel A, comparing the myosin VI nucleotide binding cleft between ADP (left) vs. rigor (right). Top panels show density maps only; bottom panels include the rigid body fit models as described in A. Density corresponding to ADP nucleotide is orange.

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  Figure 3—figure supplement 2.. Validation of ADP MDFF model. Per-residue Cα RMSD is displayed between superpositions of backbone-averaged HR MDFF models. The superposition was generated based on the Cα coordinates of the indicated full motor domains. The backbone of the first state indicated is displayed and colored. Differences of the lowest magnitude occur between ADP from 2BKI vs. ADP from 4PFO.

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  Figure 3—figure supplement 3.. Interactions composing the actomyosin VI interface in the ADP state. (A) All six actomyosin interfaces from the HR ADP MDFF model, superimposed based on the Cα coordinates of actin protomer 1 (dark-blue), as described in Figure 2A. MD, dark magenta; actin subunits, varying shades of blue. (B–F) Detailed views of interface contacts suggested by MDFF, colored as in A. EM density map is displayed on left side in transparent grey. (B) Hydrophobic interaction between MD HLH and actin SD1/SD3. (C) Milligan contact interactions between MD loop 3 and actin D-loop/SD1. (D) Electrostatic interaction between MD loop 4 and actin SD 3. (E) Interface of MD HCM loop and loop 2 with actin surface colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). (F) Salt bridge formation between the base of the MD HCM loop with actin SD1.

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  Figure 3—figure supplement 4.. Comparison of interactions composing the actomyosin VI interface between ADP and rigor. (A) All six actomyosin interfaces from the HR ADP MDFF model as described in Figure 3—figure supplement 3. MD, dark magenta; actin subunits, varying shades of blue. (B–F) Detailed views of interface contacts, colored as in A; HR rigor MDFF model, panels from Figure 2B–2F, displayed on left side for comparison. (B) Hydrophobic interaction between MD HLH and actin SD1/SD3. (C) Milligan contact interactions between MD loop 3 and actin D-loop/SD1. (D) Electrostatic interaction between MD loop 4 and actin SD 3. (E) Interface of MD HCM loop and loop two with actin surface colored by hydrophobicity from most hydrophobic (orange) to most hydrophilic (blue). (F) Salt bridge formation between the base of the MD HCM loop with actin SD1.

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  Figure 3—figure supplement 5.. Transducer rearrangement comparisons between myosins V and VI. Transducer rearrangements between ADP and Rigor. Top panels: View of the transducer from the LPF ADP MDFF model (right, purple) and LPF rigor MDFF model (left, magenta), rigid body fit into their corresponding density maps. Bottom panels: Comparison of the myosin VI (left) and myosin V (right) transducer. View of the superposition of LPF ADP MDFF (purple) and LPF rigor MDFF (magenta) models, and Myosin V ADP (EMDB 31289, dark green) and rigor (EMDB 31288, green) structures. To generate these superpositions, the ADP and rigor density maps were aligned, then their corresponding atomistic models were rigid body fit into the aligned maps.

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  Figure 3—figure supplement 6.. Milligan contact comparisons between myosins IIC, V, and VI. (A) MDFF indicates the Milligan contact cation-π interaction between R561 in the MD loop three and Y91 in the adjacent actin is absent in ADP (left) and ADP from 2BKI (right) but is established upon transition to the rigor state (middle). For all three states, all six actomyosin interfaces in the corresponding HR MDFF model are displayed superimposed on one actin subunit as described in Figure 2A. (B) Myosin V R542, analogous to R561 in myosin VI, does not form a cation-π interaction with actin Y91 in ADP (left, dark green) or rigor (right, green). Actin is displayed in dark grey. (C) Myosin IIC (light pink, PDB 5JLH) Milligan contact does not form a cation-π interaction with (non-muscle) actin Y90 (grey).

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  Figure 4.. The converter adopts the post-power stroke conformation in the ADP state accompanied by switch II closure. (A) View of the crystal structure of the PiR state (orange, 4PFO) superimposed on the LPF ADP MDFF model (dark magenta) based on the Cα coordinates of the full motor domain. Actin density is displayed in light grey. (B) Comparison of swII orientation between the PiR and ADP (left) and PiR and Rigor (right), superimposed as described in A. Density maps for ADP (left) and rigor (right) are displayed. ADP from PiR state is displayed in ball and stick representation and colored by heteroatom. (C) Comparison of swII orientations (various colors) between five states in the force generation cycle. The ADP and rigor maps were aligned, then their corresponding atomistic models were rigid body fit into the aligned maps. MDs from crystal structures were then superimposed based on the Cα coordinates of the full motor domain utilizing ADP as the reference for PiR and PPS, and rigor as the reference for Post-rigor. ADP from PiR is displayed in ball and stick representation and colored by heteroatom. (D) Magnified view of the 120° rotation of the converter and lever arm upon the transition…