Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution

Abstract

The interaction of myosin with actin filaments is the central feature of muscle contraction1 and cargo movement along actin filaments of the cytoskeleton2. The energy for these movements is generated during a complex mechanochemical reaction cycle3,4. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin5,6,7. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins8,9,10,11, a high-resolution structure of the F-actin–myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release)7 states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

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Figure 1: Structure of the ATM complex.
Figure 2: Interfaces of the ATM complex.
Figure 3: Stabilization of loop 2.
Figure 4: Comparison of PPS and rigor state and induced changes in F-actin.
Figure 5: Model of myosin binding to F-actin.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The coordinates and electron microscopy density maps have been deposited in the Protein Data Bank (PDB) under accession numbers 5JLF and 5JLH and the Electron Microscopy Data Bank (EMDB) under accession numbers EMD-8162 to EMD-8165.

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Acknowledgements

We thank O. Hofnagel for assistance in cryo sample preparation. We acknowledge R. Matadeen and S. de Carlo for image acquisition at the Netherlands Centre for Nanoscopy in Leiden. We thank R. S. Goody for reading the manuscript. This work was supported by the Max Planck Society, the European Research Council under the European Union’s Seventh Framework Program (FP7/2007-2013) (grant number 615984) (to S.R.), the Behrens-Weise foundation (to S.R.) and German Research Foundation (DFG) grant MA 1081/21-1 (to D.J.M.). J.v.d.E. is a fellow of Studienstiftung des deutschen Volkes.

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Contributions

D.J.M. and S.R. designed the project. S.M.H. and S.P.-C. purified actin, tropomyosin, and myosin constructs. D.J.M. supervised protein work. J.v.d.E. prepared specimens, recorded, analysed and processed the data, and prepared figures. S.R. managed the project. J.v.d.E. and S.R. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Stefan Raunser.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks E. Nogales, J. Löwe and A. Houdusse for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Micrographs, two-dimensional classifications and three-dimensional refinement.

ac, Representative of ~2,000 digital micrographs (a) and of 200 two-dimensional class averages of the F-actin–tropomyosin–myosin data set before (b) and after (c) three-dimensional refinement, respectively. Lower part of the micrograph is band-pass filtered to allow a better visualization of the filaments. Only filaments in rectangular boxes were chosen for refinement and bundled filaments were sorted out. d, e, Box dimension and angular distribution during three-dimensional refinement in side (d) and top (e) views. Histogram (few in blue to many in red) shows distribution of projection direction of each boxed segment relative to the three-dimensional reconstruction (grey). f, Example of two class averages out of c that show secondary structure elements. g, Fit of F-actin–myosin model to assign characteristic domains of myosin (see coloured circles and boxes). h, i, Representative of 200 class averages of the reprocessed F-actin–tropomyosin data set before (h) and after (i) three-dimensional refinement. j, k, In class averages, secondary structure elements in F-actin (green) and the coiled-coil structure of tropomyosin (yellow) are visible. Scale bars in micrograph and class averages are 50 nm and 10 nm, respectively.

Extended Data Figure 2 Resolution and model refinement of the actomyosin complex.

a, FSC curves of the cryo-EM reconstruction of the F-actin–tropomyosin–myosin data set (blue) and the reprocessed data set of F-actin–tropomyosin (red). The average resolution (FSC0.143) of the final electron density maps (central parts, green in subfigures) is estimated at 3.9 Å and 3.6 Å, respectively. Next subfigures illustrate only the actomyosin data set. b, Colour-coded local resolution of the full map and only finally refined part of the map (see Methods) estimated by ResMap43. ce, Representative regions with higher than the average resolution in the F-actin filament core (c), the average resolution at the interface (d) and lower resolution in outer myosin parts (e). f, FSC curves of the model to each half map to check for overfitting, when the model was only refined versus the first half map. Black curve shows FSC between refined model and full map, when the model was refined against the full map (see Methods). g, h, B-factor distribution of final model from low (blue) to high (red) values. The absolute value strongly depends on the sharpening factor of the map, while the distribution shows the same gradient as the local resolution in b.

Extended Data Figure 3 HLH motif bound to F-actin.

a, Front view of F-actin and the HLH motif of the L50 domain of myosin show only small changes in loop regions while helices do not alter between weak (PPS state in purple, PDB accession number 5I4E) and strong binding (rigor state in red). The D-loop is moved towards the binding interface and is stabilized (A-state in yellow, M-state in green). Arrows indicate changes and scale bar is given. b, Same view as before shows the interface of myosin and F-actin in the rigor state. One possible salt bridge is highlighted with dotted lines. Surfaces are coloured from low (white) to high (yellow) hydrophobicity. c–e, Back view of the HLH motif and the base of loop 2 bound to central (SD1, SD3) and adjacent (D-loop) F-actin subunits. Comparison of rigor (red) and PPS state (purple, PDB accession number 5I4E) shows main differences (c). Final interaction of fully bound myosin is given in d, e. Possible electrostatic interactions are indicated by dotted lines. F-actin surface is depicted per subunit colour (c), by hydrophobicity (d) or electrostatic Coulomb potential (e, −10 kcal mol−1 in red to +10 kcal mol−1 in blue). In all subfigures, coloured residue labels belong to F-actin. f, Sequence alignment of myosin (H. sapiens myosin-II, -I, -III, -V, -VI) in the region of the HLH (helix-R–loop–helix-S) motif. Important functions at the F-actin–myosin interface and roles in stabilizing these regions themselves are highlighted and labelled. Residue numbering refers to our published structure belonging to the sequence of NM-2C (depicted in bold type). Tissue localization of myosin-II is written in parentheses. We refer to the different myosin isoforms according to the nomenclature for the genes encoding the respective myosin heavy chains.

Extended Data Figure 4 Cardiomyopathy loop and disease-causing mutations.

a, Conservation of the CM-loop in the human myosin-II class is visualized as a model on F-actin (cyan) from low (white) to high (purple) conservation. b, Sequence alignment of the CM-loop region of the human myosin-II class. Important functions at the F-actin–myosin interface are highlighted and labelled. Residue numbering refers to our published structure belonging to the sequence of NM-2C (depicted in bold type). Tissue localization of myosin-II is written in parentheses. We refer to the different myosin isoforms according to the nomenclature for the genes encoding the respective myosin heavy chains. c, d, Mutations in β-cardiac myosin (MYH7) can lead to cardiomyopathies. Corresponding residues in β-cardiac myosin are illustrated with our rigor state model (c) and known mutations57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74 are listed (d). e, Table of known disease-causing mutations at the actomyosin interface56,66,69,74,75,76,77,78. Numbers in parentheses give respective residue position in our published structure of NM-2C. Localization is described in parentheses.

Extended Data Figure 5 Loop 2 and loop 3 on F-actin.

a, b, Density map (grey) corresponding to the flexible part (residues 641–661) of loop 2 (red). F-actin is shown as surface model coloured from low (white) to high (yellow) hydrophobicity (a) or electrostatic Coulomb potential (b, −10 kcal mol−1 in red to +10 kcal mol−1 in blue). Residue labels belonging to F-actin are coloured as surface colours. c, Sequence alignment of myosin (H. sapiens myosin-II, -I, -III, -V, -VI) in the region of loop 2 and helix-R of the HLH region. Important functions at the F-actin–myosin interface and in stabilizing these regions are highlighted and labelled. Residue numbering refers to our published structure belonging to the sequence of NM-2C (depicted in bold). Tissue localization of myosin-II is written in parentheses. We refer to the different myosin isoforms according to the nomenclature for the genes encoding the respective myosin heavy chains. d, e, Changes between rigor (red) and PPS state (purple) in the loop 3 region relative to the rest of lower 50-kDa domain when bound to F-actin. Movements are indicated by black arrows and scale bars are given.

Extended Data Figure 6 Sequence-dependent interaction of supporting loop with the N terminus of F-actin.

a, Surface of myosin and N terminus is depicted by electrostatic Coulomb potential (−10 kcal mol−1 in red to +10 kcal mol−1 in blue). Involved charged residues are labelled. b, Position of the proline-rich loop (supporting loop) located between relay helix and helix-R slightly differs between the PPS (purple, PDB accession number 5I4E) and rigor state (red) and shows no direct interaction with the N terminus of F-actin. Regions at the surface of SD1 are pulled to the actomyosin interface indicated by an arrow and a scale bar is given (F-actin in A-state is depicted in yellow; F-actin in M-state is depicted in cyan). c, Sequence alignment of myosin (H. sapiens myosin-II, -I, -III, -V, -VI) in the region of the supporting loop. Different lengths of the loop and a possible supporting function are given in the last column. Residue numbering refers to our published structure belonging to the sequence of NM-2C (depicted in bold). Tissue localization of myosin-II is written in parentheses. We refer to the different myosin isoforms according to the nomenclature for the genes encoding the respective myosin heavy chains. dg, Comparison of prominent properties in the supporting loop of different myosin classes (comparative models in purple) and their ability to undergo a direct interaction with the N terminus. Main differences are length of loop (numbers give absent amino acids relative to long loop) and position of the prominent positive-charged amino acid (R or K). Only an arginine or lysine sitting on the top would allow a direct interaction (eg), while a sideward-oriented arginine (d) or a short loop (e) disables or reduces a possible interaction, respectively. In addition, respective densities (d) of the cryo-EM map are displayed.

Extended Data Figure 7 Myosin-induced conformational changes in F-actin.

ac, Comparison of bare F-actin (A-state, yellow) with myosin-bound F-actin (M-state, cyan). Myosin is depicted in red. Either models (a) or representative parts of the electron density maps (b) illustrate conformational changes in F-actin (c). d, Sequence alignment of the N terminus region of human actin isoforms. Residue numbering refers to our published structure belonging to the sequence of non-muscular γ1-actin (ACTG1, depicted in bold type). To prevent confusion, the gene names instead of protein names are given. Localization is written in parentheses. e, N terminus and nucleotide binding region of F-actin undergo small changes (highlighted with arrows) through transmitted force of N terminus pulling. f, g, Close-ups of structural changes at the nucleotide binding site. h, Coordination of ADP and Mg2+ in the nucleotide binding cleft in M-state F-actin. ik, Myosin binding induces a stabilization and shifting of the C terminus towards SD1 of F-actin. C373, which was used for pyrene labelling of F-actin, is part of the C-terminal region. Scale bars are given in the subfigures.

Extended Data Figure 8 Comparison of rigor and rigor-like myosin structures.

ad, Close-ups of superimposed models from rigor-like structures (nucleotide-free myosin crystal structures in light green, PDB accession number 4PD3 (ref. 44) and 1OE9 (ref. 27)) with our rigor cryo-EM structure (red). F-actin is shown as surface model (green, cyan). Illustrated domains are labelled and coloured, while the rest of myosin is shown in grey from the rigor state model. Most regions do not show conformational differences (a, b), but the surface loops of myosin (CM-loop, loop 3 and loop 4) interacting with F-actin differ slightly in the rigor from the rigor-like structures (a, c). In contrast to the cryo-EM structure, loops at the interface (a, c) between F-actin and myosin are not always resolved in crystal structures. Major structural differences in the lever arm and converter regions are indicated by arrows and a scale bar is given (d).

Extended Data Figure 9 Different alignments of models for weak to strong binding of myosin and strut attraction to the base of loop2 promotes cleft closure.

ac, Three possible alignments of myosin in the PPS (first column, purple, PDB accession number 5I4E), Pi-release (second column, blue) and rigor (third column, red) states are illustrated with respect to F-actin. For better visualization, differences in F-actin are not shown and F-actin is only depicted in the M-state (green, cyan). The Pi-release state represents a homology model of NM-2C based on a crystal structure of myosin in the Pi-release state7 (PBD accession number 4PFO, see Methods). All models are either aligned to the U50 domain (a) or the L50 domain (b) of the rigor state. In c, the model of the Pi-release state was first aligned to the L50 domain of the rigor state. The PPS state was then aligned to the U50 domain of model of the Pi-release state. The first row in each subfigure shows changes in the U50 domain from the top (for a better visualization L50 was deleted). The second row shows the L50 domain from the bottom (U50 is transparent). Possible clashes are indicated by a yellow star (a). df, Binding mechanism of the strut, connecting L50 and U50 domains, to the stabilized base of loop 2. To illustrate the conformational changes, the respective regions in the PPS state (PPS, purple, PDB accession number 5I4E) and rigor state (RS, red) of myosin have been partly overlaid. The rest of myosin is shown in grey. L50 binds to F-actin (A-state, yellow) (d, e). The base of loop 2 is stabilized by F-actin (e) and attracts the negatively charged strut with its positive patch. This promotes the binding of the strut, shifting the equilibrium to a closed conformational state of myosin (f). Flexible parts of loop 2 are indicated as dotted lines. Lower panels show surfaces of the same regions as in the upper panels coloured by electrostatic Coulomb potential. For better visualization, the upper parts of the strut were removed. Surface of F-actin is depicted in transparent grey.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Cryo-EM structure of the ATM complex in detail

a, Cryo-EM reconstruction of F-actin (five central subunits in green and one subunit in cyan) decorated with tropomyosin (blue) and myosin in rigor state (central molecules highlighted in red). Close-up ends with the central part of the map. b, Representative part of the central core region of the F-actin filament shows better than average resolution. c, Interface between D-loop, SD1 of F-actin and HLH motif of myosin. d, Myosin CM-loop bound to SD1 and SD3 of F-actin. (MP4 9614 kb)

Model of the rigor ATM complex and subdomains in F-actin

F-actin (green, cyan) decorated with tropomyosin (blue) and myosin (red). Subdomain organisation of F-actin is shown. (MP4 8246 kb)

Domain organisation of myosin on F-actin

Domain organisation of the NM-2C head region (depicted in different colours and labelled as in Fig. 1b-d). Close-ups on HLH motif and surface loops (CM-loop, loop 3, loop 4) at the F-actin-myosin interface. (MP4 9341 kb)

F-actin-myosin interfaces in detail

a-c, Close-ups on the HLH motif (a), CM-loop (b) and loop 2 (c) of myosin bound to F-actin in rigor state. d, e, A positively charged basin is formed by loop 2, helix-W and the supporting loop of myosin to stabilize the pulled N-terminus conformation of F-actin. Myosin is shown as ribbon with residues (d) or as surface coloured by the electrostatic Coulomb potential ranging from -10 kcal mol-1 in red to +10 kcal mol-1 in blue. (MP4 7622 kb)

Model of weak to strong binding of myosin to F-actin

a, Myosin binds in a weak PPS state (purple, PDB: 5I4E) to F-actin. b, L50 rotates and binds to F-actin resulting in a stronger bound myosin (Pi-release state in blue). c, d, Finally, U50 rotates and binds to F-actin (c) obtaining the full interface of strongly bound myosin (rigor state in red) on F-actin (d). Models were aligned as shown in Extended Data Fig. 9f. For better visualization, differences in F-actin are not shown and F-actin is depicted only in the M-state (green, cyan). (MP4 5344 kb)

Model of myosin-binding to F-actin in detail

a, Initial binding of myosin (purple) to F-actin (yellow). b, L50 rotates and binds to F-actin. c, The base of loop 2 (red) is stabilized by hydrophobic interactions with F-actin. d, It also interacts with the negatively charged N-terminus of F-actin inducing its pulling and ordering (A-state in yellow, M-state in cyan). L50 is in its final rigor state conformation. e, Negatively charged strut gets attracted to the positively charged patch of the base of loop 2. U50 rotates and binds to F-actin resulting in the interface observed in the rigor state (red). (MP4 6017 kb)

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Ecken, J., Heissler, S., Pathan-Chhatbar, S. et al. Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution. Nature 534, 724–728 (2016). https://doi.org/10.1038/nature18295

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