scholarly journals The structural basis of muscle contraction

2000 ◽  
Vol 355 (1396) ◽  
pp. 419-431 ◽  
Author(s):  
Kenneth C. Holmes ◽  
Michael A. Geeves
Keyword(s):  
Active Site ◽  
Smooth Muscle Actin ◽  
Actin Binding ◽  
Power Stroke ◽  
Structural Basis ◽  
Myosin Filament ◽  
Muscle Actin ◽  
Phosphate Release ◽  
Cross Bridge ◽  
Lever Arm

The myosin cross–bridge exists in two conformations, which differ in the orientation of a long lever arm. Since the lever arm undergoes a 60° rotation between the two conformations, which would lead to a displacement of the myosin filament of about 11nm, the transition between these two states has been associated with the elementary ‘power stroke’ of muscle. Moreover, this rotation is coupled with changes in the active site (CLOSED to OPEN), which probably enable phosphate release. The transition CLOSED to OPEN appears to be brought about by actin binding. However, kinetics shows that the binding of myosin to actin is a two–step process which affects both ATP and ADP affinity and vice versa. The structural basis of these effects is only partially explained by the presently known conformers of myosin. Therefore, additional states of the myosin cross–bridge should exist. Indeed, cryoelectron microscopy has revealed other angles of the lever arm induced by ADP binding to a smooth muscle actin–myosin complex.

scholarly journals FRET and optical trapping reveal mechanisms of actin activation of the power stroke and phosphate release in myosin V

2020 ◽  
Vol 295 (51) ◽  
pp. 17383-17397 ◽  
Author(s):  
Laura K. Gunther ◽  
John A. Rohde ◽  
Wanjian Tang ◽  
Joseph A. Cirilo ◽  
Christopher P. Marang ◽  
...  
Keyword(s):  
Active Site ◽  
Maximum Rate ◽  
Optical Trapping ◽  
Structural Changes ◽  
Nucleotide Binding ◽  
Power Stroke ◽  
Myosin V ◽  
Phosphate Release ◽  
Lever Arm ◽  
Binding Regions

Myosins generate force and motion by precisely coordinating their mechanical and chemical cycles, but the nature and timing of this coordination remains controversial. We utilized a FRET approach to examine the kinetics of structural changes in the force-generating lever arm in myosin V. We directly compared the FRET results with single-molecule mechanical events examined by optical trapping. We introduced a mutation (S217A) in the conserved switch I region of the active site to examine how myosin couples structural changes in the actin- and nucleotide-binding regions with force generation. Specifically, S217A enhanced the maximum rate of lever arm priming (recovery stroke) while slowing ATP hydrolysis, demonstrating that it uncouples these two steps. We determined that the mutation dramatically slows both actin-induced rotation of the lever arm (power stroke) and phosphate release (≥10-fold), whereas our simulations suggest that the maximum rate of both steps is unchanged by the mutation. Time-resolved FRET revealed that the structure of the pre– and post–power stroke conformations and mole fractions of these conformations were not altered by the mutation. Optical trapping results demonstrated that S217A does not dramatically alter unitary displacements or slow the working stroke rate constant, consistent with the mutation disrupting an actin-induced conformational change prior to the power stroke. We propose that communication between the actin- and nucleotide-binding regions of myosin assures a proper actin-binding interface and active site have formed before producing a power stroke. Variability in this coupling is likely crucial for mediating motor-based functions such as muscle contraction and intracellular transport.

Novel configuration of a myosin II transient intermediate analogue revealed by quick-freeze deep-etch replica electron microscopy

2013 ◽  
Vol 450 (1) ◽  
pp. 23-35 ◽  
Author(s):  
Yoshitaka Kimori ◽  
Norio Baba ◽  
Eisaku Katayama
Keyword(s):  
Electron Microscopy ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Power Stroke ◽  
Structural Analogue ◽  
Unusual Structure ◽  
Mapping Procedure ◽  
Cross Bridge ◽  
Lever Arm ◽  
Transient Intermediate

In the present paper, we described our attempt to characterize the rough three-dimensional features of the structural analogue of the key intermediate of myosin's cross-bridge cycle. Using quick-freeze deep-etch replica electron microscopy, we observed that actin-attached myosin during in vitro sliding was bent superficially as postulated by the conventional hypothesis, but in the opposite direction of the putative pre-power-stroke configuration, as for ADP·Vi (inorganic vanadate)-bound myosin. We searched for the conformational species with a similar appearance and found that SH1–SH2 (thiols 1 and 2)-cross-linked myosin is a good candidate. To characterize such small asymmetric structures, we employed a new pattern-recognition procedure that accommodates the metal-replicated samples. In this method, the best-matched views of the target microscopic images were selected from a comprehensive set of images simulated from known atomic co-ordinates of relevant proteins. Together with effective morphological filtering, we could define the conformational species and the view angles of the catalytic domain and the lever arm cropped from averaged images of disulfide-cross-linked myosin. Whereas the catalytic domain of the new conformer closely resembled the pPDM (N,N′-p-phenylenedimaleimide)-treated, but SH2 Lys705-cross-linked, structure (PDB code 1L2O), a minor product of the same cross-linking reaction, the lever arm projected differently. Using separately determined view angles of the catalytic domain and the lever arm, we built a model of disulfide-cross-linked myosin. Further combination with the ‘displacement-mapping’ procedure enabled us to reconstruct the global three-dimensional envelope of the unusual structure whose lever arm orientation is compatible with our reports on the actin-sliding cross-bridge structure. Assuming this conformer as the structural analogue of the transient intermediate during actin sliding, the power stroke of the lever arm might accompany the reversal of the disorganized SH1 helix.

scholarly journals Switch-1 Instability at the Active Site Decouples ATP Hydrolysis from Force Generation in Myosin II

2020 ◽  
Author(s):  
Benjamin C. Walker ◽  
Claire E. Walczak ◽  
Jared C. Cochran
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Slight Modification ◽  
Divalent Metal ◽  
Force Generation ◽  
Single Atom ◽  
Actin Binding ◽  
Power Stroke ◽  
Tight Coupling

AbstractMyosin active site elements (i.e. switch-1) bind both ATP and a divalent metal to coordinate ATP hydrolysis. ATP hydrolysis at the active site is linked via allosteric communication to the actin polymer binding site and lever arm movement, thus coupling the free energy of ATP hydrolysis to force generation. How active site motifs are functionally linked to actin binding and the power stroke is still poorly understood. We hypothesize that destabilizing switch-1 movement at the active site will negatively affect the tight coupling of ATP hydrolysis to force production. Using a metal-switch system, we tested the effect of interfering with switch-1 coordination of the divalent metal cofactor on force generation. We found that while ATPase activity increased, motility was inhibited. Our results demonstrate that a single atom change that affects the switch-1 interaction with the divalent metal directly regulates actin binding and force generation. Even slight modification of the switch-1 divalent metal coordination can decouple ATP hydrolysis from motility. Switch-1 movement is therefore critical for both structural communication with the actin binding site, as well as coupling the energy of ATP hydrolysis to force generation.

scholarly journals Direct real-time detection of the structural and biochemical events in the myosin power stroke

2015 ◽  
Vol 112 (46) ◽  
pp. 14272-14277 ◽  
Author(s):  
Joseph M. Muretta ◽  
John A. Rohde ◽  
Daniel O. Johnsrud ◽  
Sinziana Cornea ◽  
David D. Thomas
Keyword(s):  
Force Generation ◽  
Actin Binding ◽  
Power Stroke ◽  
Transient Time ◽  
Time Resolved ◽  
Phosphate Release ◽  
Biochemical Kinetics ◽  
Temporal Coupling ◽  
Muscle Myosin ◽  
Kinetics Of

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)2FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals Dynamics of actomyosin interactions in relation to the cross-bridge cycle

2004 ◽  
Vol 359 (1452) ◽  
pp. 1843-1855 ◽  
Author(s):  
K. C. Holmes ◽  
D. R. Trentham ◽  
R. Simmons ◽  
Wei Zeng ◽  
Paul B. Conibear ◽  
...  
Keyword(s):  
Motor Domain ◽  
Actin Binding ◽  
Kinetic Measurements ◽  
Product Release ◽  
Myosin Motor ◽  
Cross Bridge ◽  
Lever Arm ◽  
Arm Position ◽  
Myosin Motor Domain ◽  
The Cross

Transient kinetic measurements of the actomyosin ATPase provided the basis of the Lymn–Taylor model for the cross–bridge cycle, which underpins current models of contraction. Following the determination of the structure of the myosin motor domain, it has been possible to introduce probes at defined sites and resolve the steps in more detail. Probes have been introduced in the Dicytostelium myosin II motor domain via three routes: (i) single tryptophan residues at strategic locations throughout the motor domain; (ii) green fluorescent protein fusions at the N and C termini; and (iii) labelled cysteine residues engineered across the actin–binding cleft. These studies are interpreted with reference to motor domain crystal structures and suggest that the tryptophan (W501) in the relay loop senses the lever arm position, which is controlled by the switch 2 open–to–closed transition at the active site. Actin has little effect on this process per se . A mechanism of product release is proposed in which actin has an indirect effect on the switch 2 and lever arm position to achieve mechanochemical coupling. Switch 1 closing appears to be a key step in the nucleotide–induced actin dissociation, while its opening is required for the subsequent activation of product release. This process has been probed with F239W and F242W substitutions in the switch 1 loop. The E706K mutation in skeletal myosin IIa is associated with a human myopathy. To simulate this disease we investigated the homologous mutation, E683K, in the Dictyostelium myosin motor domain.

scholarly journals The structure of the rigor complex and its implications for the power stroke

2004 ◽  
Vol 359 (1452) ◽  
pp. 1819-1828 ◽  
Author(s):  
K. C. Holmes ◽  
D. R. Trentham ◽  
R. Simmons ◽  
K. C. Holmes ◽  
R. R. Schröder ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Model System ◽  
Actin Binding ◽  
Power Stroke ◽  
Dimensional Electron ◽  
Myosin V ◽  
Cross Bridge ◽  
Dimensional Reconstruction ◽  
Skeletal Myosin

Decorated actin provides a model system for studying the strong interaction between actin and myosin. Cryo–energy–filter electron microscopy has recently yielded a 14 Å resolution map of rabbit skeletal actin decorated with chicken skeletal S1. The crystal structure of the cross–bridge from skeletal chicken myosin could not be fitted into the three–dimensional electron microscope map without some deformation. However, a newly published structure of the nucleotide–free myosin V cross–bridge, which is apparently already in the strong binding form, can be fitted into the three–dimensional reconstruction without distortion. This supports the notion that nucleotide–free myosin V is an excellent model for strongly bound myosin and allows us to describe the actin–myosin interface. In myosin V the switch 2 element is closed although the lever arm is down (post–power stroke). Therefore, it appears likely that switch 2 does not open very much during the power stroke. The myosin V structure also differs from the chicken skeletal myosin structure in the nucleotide–binding site and the degree of bending of the backbone ß–sheet. These suggest a mechanism for the control of the power stroke by strong actin binding.

scholarly journals Cardiomyopathy Mutations Impact the Power Stroke of Human Cardiac Myosin

2020 ◽  
Author(s):  
W. Tang ◽  
J. Ge ◽  
W.C. Unrath ◽  
R. Desetty ◽  
C. M. Yengo
Keyword(s):  
Cardiac Muscle ◽  
Actin Filaments ◽  
Structural Changes ◽  
Power Stroke ◽  
Contractile Dysfunction ◽  
Cardiac Myosin ◽  
Phosphate Release ◽  
Force Development ◽  
Lever Arm ◽  
Muscle Myosin

AbstractCardiac muscle contraction is driven by the molecular motor myosin that uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, while it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a Förster resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic stopped-flow measurements. We proposed that the fast phase measured with our FRET probes represents the structural change associated with rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated human cardiac muscle myosin has a slower power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the power stroke and phosphate release revealed that the power stroke occurs before phosphate release, and the two steps are tightly coupled. We speculate that the slower power stroke rate constant in cardiac myosin may correlate with the slower force development and/or unique thin filament activation properties in cardiac muscle. Additionally, we demonstrated that HCM (R723G) and DCM (F764L) associated mutations both reduced actin-activation of the power stroke in M2β-S1. We also demonstrate that both mutations decrease ensemble force development in the loaded in vitro motility assay. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.SignificanceMutations in human beta-cardiac myosin are known to cause various forms of heart disease, while it is unclear how the mutations lead to contractile dysfunction and pathogenic remodeling of the heart. In this study, we investigated two mutations with opposing phenotypes and examined their impact on ATPase cycle kinetics, structural changes associated with the myosin power stroke, and ability to slide actin filaments in the presence of load. We found that both mutations impair the myosin power stroke and other key kinetic steps as well as the ability to produce ensemble force. Thus, our results provide a structural basis for how mutations disrupt molecular level contractile dysfunction.

Visualizing myosin's power stroke in muscle contraction

2000 ◽  
Vol 113 (20) ◽  
pp. 3551-3562
Author(s):  
M.C. Reedy
Keyword(s):  
Catalytic Domain ◽  
Insect Flight ◽  
Actin Binding ◽  
Power Stroke ◽  
X Ray Diffraction ◽  
Motor Action ◽  
Arm Swing ◽  
Lever Arm ◽  
Total Interaction

The long-standing swinging crossbridge or lever arm hypothesis for the motor action of myosin heads finds support in recent results from 3-D tomograms of insect flight muscle (IFM) fast frozen during active contraction and from both fluorescence polarization and X-ray diffraction during rapid stretches or releases of isometrically contracting fibers. The latter provide direct evidence for lever arm movements synchronous with force changes. Rebuilding the atomic model of nucleotide-free subfragment 1 (S1) to fit fast-frozen, active IFM crossbridges suggests a two-stage power stroke in which the catalytic domain rolls on actin from weak to strong binding; this is followed by a 5-nm lever arm swing of the light chain domain, which gives a total interaction distance of approx. 12 nm. Comparison of S1 crystal structures with in situ myosin heads suggests that actin binding may be necessary in order to view the full repertoire of myosin motor action. The differing positions of the catalytic domains of actin-attached myosin heads in contracting IFM suggest that both the actin-myosin binding energy and the hydrolysis of ATP may be used to cock the crossbridge and drive the power stroke.

High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

1992 ◽  
Vol 50 (1) ◽  
pp. 512-513
Author(s):  
J. Jakana ◽  
M.F. Schmid ◽  
P. Matsudaira ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Binding Proteins ◽  
Actin Binding ◽  
Eukaryotic Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Actin Bundle ◽  
Actin Bundles ◽  
3 Dimensional ◽  
Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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