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Cited by 27Cardiomyopathy and Myosin StudiesProtein Structure and DynamicsAdvanced Electron Microscopy Techniques and Applications

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

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Doran, Matthew H., Rynkiewicz, Michael J., Rasicci, David, Bodt, Skylar M.L., Barry, Meaghan E., Bullitt, Esther, Yengo, Christopher M., Moore, Jeffrey R., Lehman, William·2023·Journal of General Physiology
DOI 10.1085/jgp.202213267PMC PMC9859928

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Abstract

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.

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Figures

Figure 1. Conformational changes in the myosin motor domain produce force on actin. Structural organization of the myosin motor. (A) Ribbon diagram of the myosin S1 motor domain displayed in two orientations. The secondary structure of the molecule is colored according to the subdomains of the protein, including the upper 50 kD domain (U50, dark purple), the lower 50 kD domain (L50, light purple), the N-terminal domain (blue), converter (cyan), lever arm (magenta), and the transducer region (light blue). The nucleotide-binding site is marked by a space-filling model of ADP/Mg. This figure was adapted from Doran and Lehman (2021) and uses the coordinates deposited in PDB ID 5H53. (B) Schematic of the myosin force generating mechanism. β-cardiac myosin II undergoes conformational changes that are paired to actin binding and nucleotide-hydrolysis product release steps in order to generate the requisite force that results in cardiac muscle contraction. This figure describes each step of the cycle and the rearrangements of the myosin head, including cleft closure and the movement of the lever arm during the powerstroke. Each subdomain of the molecule is colored as in A, while the force generating steps are shaded red. In order to provide clarity, the phosphate release step, which occurs after hydrolysis and before the first step of the powerstroke, was not included in this figure. The step we focus on in this paper, ADP-release, is outlined in the dashed box.
Figure 1. Conformational changes in the myosin motor domain produce force on actin. Structural organization of the myosin motor. (A) Ribbon diagram of the myosin S1 motor domain displayed in two orientations. The secondary structure of the molecule is colored according to the subdomains of the protein, including the upper 50 kD domain (U50, dark purple), the lower 50 kD domain (L50, light purple), the N-terminal domain (blue), converter (cyan), lever arm (magenta), and the transducer region (light blue). The nucleotide-binding site is marked by a space-filling model of ADP/Mg. This figure was adapted from Doran and Lehman (2021) and uses the coordinates deposited in PDB ID 5H53. (B) Schematic of the myosin force generating mechanism. β-cardiac myosin II undergoes conformational changes that are paired to actin binding and nucleotide-hydrolysis product release steps in order to generate the requisite force that results in cardiac muscle contraction. This figure describes each step of the cycle and the rearrangements of the myosin head, including cleft closure and the movement of the lever arm during the powerstroke. Each subdomain of the molecule is colored as in A, while the force generating steps are shaded red. In order to provide clarity, the phosphate release step, which occurs after hydrolysis and before the first step of the powerstroke, was not included in this figure. The step we focus on in this paper, ADP-release, is outlined in the dashed box.CC BY-NC-SA · PubMed Central
Figure 2. Composite cryo-EM reconstructions of the human cardiac actin–β-myosin–tropomyosin complex. (A) Top: Composite cryo-EM map of the rigor actin–β-cardiac myosin II–tropomyosin complex. These reconstructions were created by combining the central portion of the actin-tropomyosin from the helical reconstructions and the S1 domain of myosin, including the essential light chain (ELC) from focused refinements. This figure was rendered in Chimerax using the surface zone tool (Pettersen et al., 2021). Bottom: Atomic model of the complex in the same orientation as the reconstruction above. (B). Top: Mg2+·ADP-bound complex composite map. The rendering of the ADP-bound complex was completed in the same fashion as the rigor complex. Bottom: Atomic model of the rigor complex in the same orientation as the reconstruction. The nucleotide is also indicated in both the reconstruction and model.
Figure 2. Composite cryo-EM reconstructions of the human cardiac actin–β-myosin–tropomyosin complex. (A) Top: Composite cryo-EM map of the rigor actin–β-cardiac myosin II–tropomyosin complex. These reconstructions were created by combining the central portion of the actin-tropomyosin from the helical reconstructions and the S1 domain of myosin, including the essential light chain (ELC) from focused refinements. This figure was rendered in Chimerax using the surface zone tool (Pettersen et al., 2021). Bottom: Atomic model of the complex in the same orientation as the reconstruction above. (B). Top: Mg2+·ADP-bound complex composite map. The rendering of the ADP-bound complex was completed in the same fashion as the rigor complex. Bottom: Atomic model of the rigor complex in the same orientation as the reconstruction. The nucleotide is also indicated in both the reconstruction and model.CC BY-NC-SA · PubMed Central
Figure S1. Cryo-EM image processing for the rigor complex. (A) Representative cryo-EM micrograph of the human cardiac actin–tropomyosin–myosin rigor complex, taken from a dataset of 3,903. Scale bar: 50 nm. Adjacent to the micrograph are representative 2-D class averages of the complex, where the actin, tropomyosin, and myosin can be seen in more detail. Scale bar: 5 nm. (B) Flowchart describing the cryo-EM data processing scheme to determine the helically averaged human cardiac actin–tropomyosin–myosin rigor complex. This flowchart also includes the focused classification scheme that improved the reconstruction of the myosin–essential light chain complex. Briefly, after generating 2-D class averages from manually picked particles, autopicking produced 1,045,903 segments. Subsequent 2-D and 3-D classifications identified the best segments for use in 3-D auto-refine. Further refinements produced a final helically averaged map to 3.4 Å resolution. Focused classification produced the final 3.8 Å reconstruction of the myosin–essential light-chain complex.
Figure S1. Cryo-EM image processing for the rigor complex. (A) Representative cryo-EM micrograph of the human cardiac actin–tropomyosin–myosin rigor complex, taken from a dataset of 3,903. Scale bar: 50 nm. Adjacent to the micrograph are representative 2-D class averages of the complex, where the actin, tropomyosin, and myosin can be seen in more detail. Scale bar: 5 nm. (B) Flowchart describing the cryo-EM data processing scheme to determine the helically averaged human cardiac actin–tropomyosin–myosin rigor complex. This flowchart also includes the focused classification scheme that improved the reconstruction of the myosin–essential light chain complex. Briefly, after generating 2-D class averages from manually picked particles, autopicking produced 1,045,903 segments. Subsequent 2-D and 3-D classifications identified the best segments for use in 3-D auto-refine. Further refinements produced a final helically averaged map to 3.4 Å resolution. Focused classification produced the final 3.8 Å reconstruction of the myosin–essential light-chain complex.CC BY-NC-SA · PubMed Central
Figure S2. Cryo-EM image processing for ADP–Mg2+ complex. (A) Representative cryo-EM micrograph of the human cardiac actin–tropomyosin–myosin complex bound to ADP-Mg2+, taken from a dataset of 3,961. Scale bar: 50 nm. Adjacent to the micrograph are representative 2-D class averages of the complex, where the actin, tropomyosin, and myosin can be seen in more detail. Scale bar: 5 nm. (B) Flowchart describing the cryo-EM data processing scheme to determine the helically averaged human cardiac actin–tropomyosin–myosin complex bound to ADP. This flowchart also includes the focused classification scheme that improved the reconstruction of the myosin–essential light-chain complex. Briefly, after generating 2-D class averages from manually picked particles, autopicking produced 628,787 segments. Subsequent 2-D and 3-D classifications identified the best segments for use in 3-D auto-refine. Further refinements produced a final helically averaged map to 3.3 Å resolution. Focused classification produced the final 3.8 Å reconstruction of the myosin–essential light-chain complex bound to ADP.
Figure S2. Cryo-EM image processing for ADP–Mg2+ complex. (A) Representative cryo-EM micrograph of the human cardiac actin–tropomyosin–myosin complex bound to ADP-Mg2+, taken from a dataset of 3,961. Scale bar: 50 nm. Adjacent to the micrograph are representative 2-D class averages of the complex, where the actin, tropomyosin, and myosin can be seen in more detail. Scale bar: 5 nm. (B) Flowchart describing the cryo-EM data processing scheme to determine the helically averaged human cardiac actin–tropomyosin–myosin complex bound to ADP. This flowchart also includes the focused classification scheme that improved the reconstruction of the myosin–essential light-chain complex. Briefly, after generating 2-D class averages from manually picked particles, autopicking produced 628,787 segments. Subsequent 2-D and 3-D classifications identified the best segments for use in 3-D auto-refine. Further refinements produced a final helically averaged map to 3.3 Å resolution. Focused classification produced the final 3.8 Å reconstruction of the myosin–essential light-chain complex bound to ADP.CC BY-NC-SA · PubMed Central
Figure S3. Local resolution and map quality. (A) The FSC curve and local resolution estimations calculated in RELION for the indicated helical reconstructions. These plots show that despite the high global resolution (as measured with the FSC curve), the local resolution of the reconstruction varies. Both reconstructions contain high resolutions toward the interior of the maps, while the resolution decreases radially from the actin axis. Below these are map-to-model comparisons for the helical reconstruction in the actin and myosin regions. (B) FSC curves and local resolution estimations calculated in RELION for the indicated single-particle reconstructions. From these plots, it is observed that the local resolution of the single-particle reconstructions does not deviate as much as the helical reconstructions. These focused refinements allowed us to visualize the light chain at the distal regions of the myosin motor. Below these are map-to-model comparisons of the indicated myosin region.
Figure S3. Local resolution and map quality. (A) The FSC curve and local resolution estimations calculated in RELION for the indicated helical reconstructions. These plots show that despite the high global resolution (as measured with the FSC curve), the local resolution of the reconstruction varies. Both reconstructions contain high resolutions toward the interior of the maps, while the resolution decreases radially from the actin axis. Below these are map-to-model comparisons for the helical reconstruction in the actin and myosin regions. (B) FSC curves and local resolution estimations calculated in RELION for the indicated single-particle reconstructions. From these plots, it is observed that the local resolution of the single-particle reconstructions does not deviate as much as the helical reconstructions. These focused refinements allowed us to visualize the light chain at the distal regions of the myosin motor. Below these are map-to-model comparisons of the indicated myosin region.CC BY-NC-SA · PubMed Central
Figure 3. Coordination of the ADP-Mg2+ and the nucleotide-binding site differences between ADP-bound and rigor. (A) View of the nucleotide-binding pocket and the placement of the Mg2+·ADP within it. The density of the nucleotide from the single particle focused refinement is shown in grey. Side chains that are involved in the coordination and binding of the nucleotide are displayed around the ADP. (B) Ligplot diagram detailing the interaction between the myosin nucleotide-binding site and nucleotide. Hydrogen bonds are shown in green, while hydrophobic contacts are shown by red semi-circles. The orientation of the nucleotide is similar to A. (C) Comparison of the nucleotide-binding site in the rigor complex (orchid) versus the Mg2+·ADP-bound complex (gold). The changes of each part of the nucleotide pocket are displayed including the movement of the HF helix and P-loop, switch I, the purine-binding loop, the N-terminus, and loop 673–686. These comparisons were created by aligning the actin filament of the helical models and overlaying the single particle S1 models on their helical counterparts using the matchmaker command in ChimeraX.
Figure 3. Coordination of the ADP-Mg2+ and the nucleotide-binding site differences between ADP-bound and rigor. (A) View of the nucleotide-binding pocket and the placement of the Mg2+·ADP within it. The density of the nucleotide from the single particle focused refinement is shown in grey. Side chains that are involved in the coordination and binding of the nucleotide are displayed around the ADP. (B) Ligplot diagram detailing the interaction between the myosin nucleotide-binding site and nucleotide. Hydrogen bonds are shown in green, while hydrophobic contacts are shown by red semi-circles. The orientation of the nucleotide is similar to A. (C) Comparison of the nucleotide-binding site in the rigor complex (orchid) versus the Mg2+·ADP-bound complex (gold). The changes of each part of the nucleotide pocket are displayed including the movement of the HF helix and P-loop, switch I, the purine-binding loop, the N-terminus, and loop 673–686. These comparisons were created by aligning the actin filament of the helical models and overlaying the single particle S1 models on their helical counterparts using the matchmaker command in ChimeraX.CC BY-NC-SA · PubMed Central
Figure 4. Conformational changes in the motor domain associated with ADP-Mg2+ release. Comparisons of the nucleotide-free rigor form and Mg2+·ADP-bound complex reveal conformational changes associated with ADP release. (A) Comparison of the S1 motor domains of the nucleotide free-rigor form (orchid) and the Mg2+·ADP-bound complex are shown in two different orientations. We highlight significant portions of the motor domain that change from between the models including the converter domain, relay helix, N-terminus, and the lever arm. (B) Individual panels show the conformational differences in more detail for the specified regions. Small differences at the nucleotide-binding site are linked to these larger movements of subdomains. The converter domain change was measured from the α-carbon of residue S747. The lever arm rotation was measured using the axes, planes, and ellipsoids tool in Chimera. The axes used in the calculation were defined as the last 13-resiudes of the lever arm. The relay helix change was measured by the change in the α-carbon of E504.
Figure 4. Conformational changes in the motor domain associated with ADP-Mg2+ release. Comparisons of the nucleotide-free rigor form and Mg2+·ADP-bound complex reveal conformational changes associated with ADP release. (A) Comparison of the S1 motor domains of the nucleotide free-rigor form (orchid) and the Mg2+·ADP-bound complex are shown in two different orientations. We highlight significant portions of the motor domain that change from between the models including the converter domain, relay helix, N-terminus, and the lever arm. (B) Individual panels show the conformational differences in more detail for the specified regions. Small differences at the nucleotide-binding site are linked to these larger movements of subdomains. The converter domain change was measured from the α-carbon of residue S747. The lever arm rotation was measured using the axes, planes, and ellipsoids tool in Chimera. The axes used in the calculation were defined as the last 13-resiudes of the lever arm. The relay helix change was measured by the change in the α-carbon of E504.CC BY-NC-SA · PubMed Central
Figure S4. Comparing the actomyosin interface of the rigor and ADP-bound forms. (A) Model of the actin, tropomyosin, and myosin complexes. The ADP-bound form (gold) and rigor form (orcid) are overlaid in order to show the similarities of the models at the actomyosin interface. The regions of the actomyosin interface are boxed. (B) Regions of the actomyosin interface indicated from A are enlarged. The interacting residues from the CM loop, loop 4, and the helix-loop-helix motif in both myosin models are shown overlaid. It is clear from these comparisons that the interface is conserved despite the nucleotide bound to the active site.
Figure S4. Comparing the actomyosin interface of the rigor and ADP-bound forms. (A) Model of the actin, tropomyosin, and myosin complexes. The ADP-bound form (gold) and rigor form (orcid) are overlaid in order to show the similarities of the models at the actomyosin interface. The regions of the actomyosin interface are boxed. (B) Regions of the actomyosin interface indicated from A are enlarged. The interacting residues from the CM loop, loop 4, and the helix-loop-helix motif in both myosin models are shown overlaid. It is clear from these comparisons that the interface is conserved despite the nucleotide bound to the active site.CC BY-NC-SA · PubMed Central
Figure S5. Improvements from focused refinements. (A) Improvements in density from the helical to single-particle reconstruction for the rigor and ADP-bound complexes. The helically averaged reconstruction for both complexes is shown in grey, while the focused refinements are overlayed colored orchid (rigor) and yellow (ADP-bound). (B) Improvements to specific regions of the motor domain, including the lever arm and converter as well as the essential light chain.
Figure S5. Improvements from focused refinements. (A) Improvements in density from the helical to single-particle reconstruction for the rigor and ADP-bound complexes. The helically averaged reconstruction for both complexes is shown in grey, while the focused refinements are overlayed colored orchid (rigor) and yellow (ADP-bound). (B) Improvements to specific regions of the motor domain, including the lever arm and converter as well as the essential light chain.CC BY-NC-SA · PubMed Central
Figure 5. Cardiomyopathy mutations occur in regions involved in ADP release. (A) Cartoon diagram of the nucleotide-free β-cardiac myosin II S1 domain highlighting the α-carbon of residues that are known cardiomyopathy-causing mutations. Spheres indicate loci for mutations leading to hypertrophic cardiomyopathy (red), dilated cardiomyopathy (green), or both HCM and DCM (yellow). Each residue was found using the ClinVar archive filtering residues that are known to be pathogenic. (B) Shown is the location of myosin residue F764, which sits at the interface between the converter domain and the relay helix. The mutation F764 L is known to lead to DCM. (C) Shown is the location of the residue R712, which also sits at the interface between the converter domain and the relay helix. Mutations at this site are known to cause HCM. A cluster of mutations are fund in the converter domain, making it a hotspot of mutations.
Figure 5. Cardiomyopathy mutations occur in regions involved in ADP release. (A) Cartoon diagram of the nucleotide-free β-cardiac myosin II S1 domain highlighting the α-carbon of residues that are known cardiomyopathy-causing mutations. Spheres indicate loci for mutations leading to hypertrophic cardiomyopathy (red), dilated cardiomyopathy (green), or both HCM and DCM (yellow). Each residue was found using the ClinVar archive filtering residues that are known to be pathogenic. (B) Shown is the location of myosin residue F764, which sits at the interface between the converter domain and the relay helix. The mutation F764 L is known to lead to DCM. (C) Shown is the location of the residue R712, which also sits at the interface between the converter domain and the relay helix. Mutations at this site are known to cause HCM. A cluster of mutations are fund in the converter domain, making it a hotspot of mutations.CC BY-NC-SA · PubMed Central

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Authors
Doran, M. H., Rynkiewicz, M. J., Rasicci, D., Bodt, S. M. L., Barry, M. E., Bullitt, E., Yengo, C. M., Moore, J. R., & Lehman, W.
Affiliations
  • Boston University (US)
  • Pennsylvania State University (US)
  • University of Massachusetts Lowell (US)
Corresponding author
Doran, Matthew H.
ORCID
Venue
Journal of General Physiology
Publisher
Rockefeller University Press
Published
2023-01-12
Volume
155
Issue
3
ISSN
  • 0022-1295 (ISSN-L)
  • 1540-7748
DOI
10.1085/jgp.202213267
PMC
PMC9859928
OpenAlex
W4315754328
Cited by
27
References
66
Citations by year
2026: 10 · 2025: 8 · 2024: 7 · 2023: 2
FWCI
2.96
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of General Physiology (publishedVersion, cc-by-nc-sa)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nd)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health (R01HL036153, R01HL127699-03, T32HL007969, S10RR25434, U24 GM129541)
  • Boston University
Topics
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiomyopathy and Myosin Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
Keywords
cardiac myosin, ADP release, essential light chain, nucleotide binding site, force sensing, afterload, cryo-EM structure
MeSH terms
Actins, Humans, Actin Cytoskeleton, Myosins, Tropomyosin, Cardiac Myosins

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Doran, M. H., Rynkiewicz, M. J., Rasicci, D., Bodt, S. M. L., Barry, M. E., Bullitt, E., Yengo, C. M., Moore, J. R., & Lehman, W. (2023). Conformational changes linked to ADP release from human cardiac myosin bound to actin-tropomyosin. Journal of General Physiology. https://doi.org/10.1085/jgp.202213267

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