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Is Characterized by Continued Attachment of Myosin Heads to Actin Filaments Due to a Lack of Atp

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Motor Proteins

In Cell Biology (Third Edition), 2017

Transduction of Chemical Energy Into Molecular Motion

Myosin heads produce force during the transition from the AMDP state to the AMD and AM states. Production of force at this step makes sense for two reasons: First, the large free-energy difference between AMDP and AMD provides sufficient energy to produce force; second, the force-producing AMD and AM intermediates bind tightly to actin, so any force between the motor and the actin track is not dissipated. However, for many myosins, including skeletal muscle myosin, these force-producing states occupy a small fraction of the whole ATPase cycle. The fraction of the time in force producing states is called the duty cycle. ADP dissociates rapidly from AMD, and ATP binds rapidly to AM, dissociating myosin from the actin filament and initiating another ATPase cycle.

Fifty years of research using a combination of mechanical measurements, static atomic structures of myosin heads with various bound nucleotides, and spectroscopic observations of contracting muscle revealed the structural basis for the conversion of free energy into force: a dramatic conformational change in the orientation of the light-chain domain associated with phosphate dissociation (Fig. 36.5B).

Elegant mechanical experiments measured the size of the mechanical step produced by a myosin during one cycle of ATP hydrolysis. These experiments on live muscles first suggested that each cycle of ATP hydrolysis moves an actin filament approximately 5 to 10 nm relative to myosin. Now one may observe myosin moving single actin filaments by fluorescence microscopy. An array of myosin heads attached to a microscope slide can use ATP hydrolysis to push actin filaments over the surface (Fig. 36.6A–C). Assays with single myosin molecules show that each cycle of ATP hydrolysis can move an actin filament up to 5 to 15 nm and develop a force of about 3 to 7 piconewtons (pN) (Fig. 36.6D). At low ATP concentrations, the interval between the force-producing step and the binding of the next ATP is relatively long, so single steps can be observed.

Further insights emerged from biophysical studies of muscle and purified proteins using x-ray diffraction (see Fig. 39.11), electron microscopy, electron spin resonance spectroscopy, and fluorescence spectroscopy. These experiments showed that the light-chain domain pivots around a fulcrum, the converter subdomain within the catalytic domain, which is stationary relative to the actin filament. For example, spectroscopic probes on light chains revealed a change in orientation when muscle is activated to contract, whereas probes on the catalytic domain do not rotate. Crystal structures of myosin heads with various bound nucleotides and nucleotide analogs show that the light-chain domain can pivot up to 90 degrees (Fig. 36.4D). The light-chain domain is bent more acutely in the AMT and AMDP intermediates and pivots to a more extended orientation when phosphate dissociates (Fig. 36.3). ADP dissociation extends this rotation of some classes of myosin. Consistent with rotation of the light-chain domain producing movement, the rate of actin filament gliding in an in vitro assay is proportional to the length of the light-chain domain. The observed range of orientations of the light-chain domain relative to the catalytic domain can account for the observed step size of 10 nm for muscle myosin. Some aspects of these conformational changes and their relation to phosphate release are similar to Ras family GTPases (see Fig. 4.6).

Rotation of the light-chain domain is believed to produce movement indirectly in the sense that force-producing intermediates stretch elastic elements in the system. This mechanism is represented by a spring in Fig. 36.2. The elastic elements in the myosin-actin complex are most likely to be mainly in the myosin head, with small contributions from the actin and myosin filaments. Movement of the light-chain domain tensions the spring transiently in the AMD and AM states. Dissociation of ADP and rebinding of ATP to the AM intermediate reverts the system to the rapid equilibrium of mostly dissociated weakly bound intermediates. Any force left in the spring is lost as soon as the head dissociates from the actin filament.

The actual motion produced depends on the mechanical resistance in the system (Fig. 36.2). If both myosin and actin are fixed, elastic elements are stretched for the life of the force-producing states (AMD and AM), and the energy is lost as heat when the head dissociates. This happens when one tries to lift an immovable object. If the resistance is less than the force in the stretched elastic elements, the actin filament moves relative to myosin, as in muscle contraction. The distance moved in each step depends on the resistance, as the spring stops shortening when the forces are balanced.

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Molecular Motors and Motility

M. Irving , in Comprehensive Biophysics, 2012

Glossary

Actin

Track along which myosin heads move during muscle contraction; principal component of the thin filaments in muscle.

Bare zone

Central region of the muscle thick filament lacking myosin heads.

Chaotic relaxation

Late exponential phase of muscle relaxation accompanied by large local changes in sarcomere length.

Cross-bridge

Structure linking thick and thin filaments in muscle, formed by myosin head domains.

Eccentric contraction

State in which muscle actively resists an external stretch.

Equator

Axis of X-ray fiber diffraction pattern that is perpendicular to the fiber axis.

Isometric contraction

State in which muscle exerts force at constant length.

Isometric relaxation

Early linear phase of muscle relaxation in which sarcomere lengths remain constant.

Isotonic contraction

State in which muscle exerts a constant force.

Latency relaxation

Transient force decrease following electrical stimulation of muscle preceding active force development.

Layer line

Off-meridional X-ray reflection from a helical structure.

Light-chain domain

Region of the myosin head that contains the myosin light chains.

Meridian

Axis of X-ray fiber diffraction pattern that is parallel to the fiber axis.

M line

Transverse sarcomeric structure linking the mid-points of the muscle thick filaments.

Myosin

Muscle motor protein; principal component of the muscle thick filaments.

Myosin binding protein C

Thick filament component that modulates muscle contraction.

Myosin head

Globular domain of myosin that binds actin and hydrolyzes ATP.

Myosin tail

Long α-helical coiled-coil region of myosin that packs into the thick filament backbone.

Rigor

State of muscle fiber or actin-myosin complex in the absence of ATP.

Sarcomere

Structural and functional unit of striated muscle, about 2 μm long.

Sarcoplasmic reticulum

Intracellular vesicular system that serves as the calcium store in muscle.

Skinned muscle fiber

Muscle fiber in which the surface membrane has been removed or permeabilized.

Tetanus

Response of skeletal muscle to regular repetitive action potential stimulation.

Thick filament

Major component of the muscle sarcomere, containing myosin, 1.6 μm long.

Thin filament

Major component of the muscle sarcomere, containing actin, about 1.0 μm long.

Titin

Giant sarcomeric protein linking M and Z lines, assembly template for thick filaments and major source of the resting elasticity of muscle.

Tropomyosin

Thin filament regulatory protein in muscle that controls access to myosin-binding sites on actin.

Troponin

Calcium-binding regulatory protein in the muscle thin filaments.

Twitch

Response of skeletal muscle to single action potential stimulation.

Working stroke

Change in shape of the actin-attached myosin head that drives filament sliding or force generation in muscle.

X-ray fiber diffraction

Technique to obtain molecular structural information by diffraction of X-rays from oriented macromolecular fibers.

Z line

Transverse sarcomeric structure linking the muscle thin filaments; marks the ends of the sarcomere.

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Contractile Systems

N.V. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (Second Edition), 2015

Mechanism of Contraction: Cross-Bridge Cycling

The myosin neck structure allows the myosin heads to be in very close proximity to the actin. When the tropomyosin-mediated inhibition of contraction is reversed, the myosin heads interact with actin. Myosin binds initially via loop regions near the catalytic site, followed by progressively more extensive hydrogen bonding. In this process, a large surface area on each protein is removed from interaction with cell water during formation of the bond between them. In cases in which proteins bind without changes in conformation, such interaction would be associated with an energy change of 60–80  kJ/mol of bonds, a number roughly twice as great as that actually observed for binding of myosin to actin. Apparently, as the myosin moves into this tightly bound configuration, energy is captured in the form of deformation within the myosin head, which applies force (5–10 pico newtons) to the head's attachment to the thick filament (the neck region). Much, probably most, of this energy is available to do mechanical work as the filaments slide past one another (the power stroke illustrated schematically in Figure 19.4), with myosin ending up very firmly bound to actin. Subsequent binding of MgATP to myosin provides the energy input to alter myosin's actin binding site to a low-affinity state, permitting detachment. ATP binding lowers the affinity of myosin for actin by a factor of about 104. Hydrolysis of the ATP then occurs, with little change in the free energy of the system. Subsequent events release Pi and ADP from the ATPase site.

Figure 19.4. Schematic summary of the events of the cross-bridge cycle. At step 1, ATP binding allows the release of myosin from actin at the end of the prior power stroke. Ordinarily, ATP hydrolyzes while myosin is still attached to actin, the resulting M-ADP-Pi readily detaching from actin. In this state, myosin can reattach to actin, forming progressively stronger bonds as Pi and then ADP are released, resulting in the tightly bound rigor state. The free energy of binding of ATP to the myosin changes the conformation of its actin binding site in such a way that the affinity for actin is drastically reduced. At rest, most myosin heads will be in the M-ADP-Pi state, ready to enter stage 3 in this diagram when circumstances allow.

 [Modified from R. Cooke, Modulation of the actomyosin interaction during fatigue of skeletal muscle, Muscle Nerve 36 (2007) 756–777.]

Thus, in the cross-bridge cycle, myosin is bound with high affinity alternately to actin and to ATP, during which the ATP is hydrolyzed. Thus, the overall result is the conversion of energy of hydrolysis of ATP (about 50–60   kJ/mol under physiological conditions) to work (and heat), a process called chemomechanical transduction. The efficiency of this process in mammalian skeletal muscle is approximately 60%.

ATP hydrolysis normally occurs after transition of the actomyosin-ATP (A-M-ATP) complex to a weakly bound state. The complex A-M-ADP-Pi may remain weakly bound until dislodged by movement of the filaments. The released M-ADP-Pi has moderate affinity for actin, and upon reattachment, forming A-M-ADP-Pi, the phosphate release step occurs. This creates a state called A-M*-ADP that is the high-affinity state associated with initiating the power stroke. As the structural changes produced by increasingly tight binding to actin produce strain in the myosin head and therefore force and movement, the affinity of the ATPase site for ADP decreases, releasing the ADP. Thus, at the end of the power stroke, cross-bridges are typically in the A-M state (called the rigor state), their most tightly bound state, in which they will remain unless ATP is available to bind to the ATPase site and lower the affinity of the actin binding site. This model of events is illustrated schematically in Figure 19.4. In normal circumstances, it is almost impossible to deplete ATP to the point where a large proportion of myosin heads form rigor bonds, but it does happen in severely ischemic muscle and postmortem (rigor mortis). So long as [Ca2+]i remains high, this cycle will continue, provided that adequate [ATP] and other appropriate conditions of the internal environment are maintained.

Since hydrolysis of one ATP occurs for every (or almost every) cross-bridge cycle in each sarcomere of each myofibril, the ATP consumption associated with contraction can be enormous, up to 100 times the resting level. There must necessarily be metabolic specializations to meet this peak demand, and to do so quickly. Energy metabolism is discussed in Chapters 13 to 16 Chapter 13 Chapter 14 Chapter 15 Chapter 16 , and some muscle-specific aspects are discussed later, under "Energy Supply in Muscle."

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The Muscular System

Bruce M. Carlson MD, PhD , in The Human Body, 2019

The cross-bridge cycle begins when a myosin head (which protrudes 90 degrees from the long axis of the thick filament) with bound ADP plus an inorganic phosphate group (P i) attaches to a myosin-binding site on the actin filament. Release of Pi from the complex triggers the power stroke, during which the myosin head bends from 90 to 45 degrees. The bending of the myosin heads moves the actin filament 10–12   nm along the myosin toward the center of the shortening sarcomere, and this is the action that generates the force in muscle contraction (Fig. 5.9). ADP is then dissociated from the myosin molecule, and the myosin head (the cross-bridge) remains firmly attached to actin. This condition persists until a new molecule of ATP binds to the myosin head, causing it to be released from the actin filament. 1 Following the release of the myosin head from actin, hydrolysis of the ATP to ADP + Pi causes the myosin head to become re-cocked from the 45- to the 90-degree position, and the cross-bridge cycle begins again. During the re-cocking phase, the cross-bridges are not in contact with the actin filament, and the muscle is in the relaxed state.

Figure 5.9. The sliding filament model of muscle contraction. (A) Relationship between thick and thin filaments in a relaxed muscle fiber. (B) The same, in a contracted muscle fiber.

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Molecular Motors and Motility

M. Preller , D.J. Manstein , in Comprehensive Biophysics, 2012

4.8.1.3 Domain Topology and Structure of the Myosin Head

Figure 3 shows the structure of the myosin head fragment as a ribbon diagram and a schematic topology map indicating the subdomains and their secondary structural elements. The core of the myosin motor domain is formed by a central, seven-stranded β sheet which is surrounded by α-helices. A SH3-like β-barrel domain is positioned close to the N terminus. The function of this small domain is unknown; however, it is absent in class-1 myosins and thus appears not to be essential for motor activity. A large structural domain, that accounts for six of the seven strands of the central β sheet, is formed by residues 81 to 454 and 594 to 629. Unless otherwise stated, sequence numbering refers to the Dictyostelium discoideum myosin-2 heavy chain throughout this chapter. This domain is usually referred to as the upper 50-kDa domain (U50). A large cleft divides the upper 50-kDa domain from the lower 50-kDa domain (L50), a well-defined structural domain formed by residues 465 to 590. The actin binding region and nucleotide binding site of myosin are on opposite sides of the seven-stranded β sheet and phosphate moiety of the nucleotide at the rear of the nucleotide binding pocket. The P-loop, Switch-1, and switch-2 are located in the upper 50-kDa domain close to the apex of the large cleft. All three nucleotide binding motifs contact the phosphate moiety of the nucleotide at the rear of the nucleotide binding pocket and act as β-phosphate sensors. Structures obtained in the absence and presence of ATP analogues indicate that the switch motifs move toward each other when ATP is bound, and move away from each other in its absence. Switching between ATP and ADP states is associated with specific intramolecular movements, analogous to the nucleotide-dependent conformational transitions in G-proteins. 84 Conformational changes during the transition between different nucleotide states appear mostly to correspond to rigid-body rotations of secondary and tertiary structural elements. The motor domain can thus be regarded as consisting of communicating functional units, with substantial movement occurring in only a few residues. Residues 411 to 441 form a long helix which runs from the actin binding region at the tip of the large cleft to the fifth strand of the central β sheet. A broken helix is formed by residues 669 to 689. The broken helix is frequently referred to as the 'SH1-' and 'SH2-helix region', named for reactive cysteine residues. The SH1/SH2 region is tightly linked to the converter domain, that functions as a socket for the C-terminal light-chain binding domain. The relay helix, a long helix which emerges from the Switch-2 region, is in contact with the SH1 helix. As its name implies, the relay helix functions in communicating conformational information between the actin binding site, nucleotide binding site, and the mechanical amplifier elements in the neck region.

Figure 3. (a) Ribbon representation of the chicken myosin head fragment X-ray structure (PDB2MYS) color coded for the proteolytic subdomains. Red, N-terminal SH3-like domain; blue, U50-kDa domain; green, L50-kDa domain; gray, converter domain; purple, essential light chain; pale green, regulatory light chain; yellow, light-chain binding IQ motifs. (b) Topology map of the myosin-2 motor domain. Helices are shown as cylinders and β strands as arrows.

There are approximately 70 myosin motor domain structures deposited in the protein database (www.rcsb.org) derived from X-ray crystallography (Table 3), with most derived from Dd myosin-2. Other structures were derived from scallop (Pm and Ai), squid (Lp), chicken (Gg), or pig (Ss). Crystals were obtained under various conditions, with and without bound nucleotide or in complex with a series of nucleotide analogues. There are no structures of human myosins yet, and there are no high-resolution structures of full-length myosins or myosins in complex with actin filaments (F-actin). Despite their highly conserved features, only the motor domains of members of four myosin classes have been characterized structurally. In addition to the various structures of class-2 myosins, the structure of one myosin-1 motor domain (Protein Data Bank [PDB] 1LKX), four structures of chicken myosin-5a motor domains (PDB 1W7I, 1W7J, 1W8J, and 1OE9), and five structures of class-6 myosin motor domains (PDB 2VAS, 2VB6, 2V26, 2BKH, and 2BKI) have been solved. In other words, our understanding of structure-function relationships in the myosin and actomyosin systems is restricted to the motor domain, several more common interaction motifs in the tail region of unconventional myosins, and the neck regions of a small selection of myosin superfamily members. However, hybrid approaches, combining high-resolution structural information with results obtained using other biophysical and computational techniques, have produced insights into many aspects of the molecular mechanisms governing myosin function). 11,85

Table 3. Available myosin X-ray structures in the Protein Data Bank (PDB) database (www.rcsb.org)

Myosin class Organism Ligand 1 (active site) Ligand 2 State (ATPase cycle) Cleft Active site Lever arm position Resolution (Å) No. of amino acids PDB ID Ref.
Motor domain
1 Dd Mg·ADP·VO4 — Prepower stroke Partially closed Closed Up 3.0 697 1LKX 118
2 Dd Mg·ADP — Postrigor Open Open Down 2.3 776 1JWY 179
2 Dd Mg·ADP — Postrigor Open Open Down 2.3 776 1JX2 179
2 Dd Mg·ADP/R238E — Postrigor Open Open Down 2.8 1010 1G8X 116b
2 Dd — — Postrigor Open Open Down 2.1 761 1FMV 180
2 Dd Mg·ATP — Postrigor Open Open Down 2.15 761 1FMW 180
2 Dd Mg·m-NphAE·BeF3 — Postrigor Open Open Down 2.0 761 1D0X 181
2 Dd Mg·o-NphAE·BeF3 — Postrigor Open Open Down 2.0 761 1D0Y 181
2 Dd Mg·p-NphAE·BeF3 — Postrigor Open Open Down 2.0 761 1D0Z 187
2 Dd Mg·o, p-NphAE·BeF3 — Postrigor Open Open Down 2.0 761 1D1A 181
2 Dd Mg·o, p-NPhAP·BeF3 — Postrigor Open Open Down 2.0 761 1D1B 181
2 Dd Mg·N-methyl-NphAE·BeF3 — Postrigor Open Open Down 2.3 761 1D1C 181
2 Dd Mg·MNT — Postrigor Open Open Down 1.9 762 1LVK 182
2 Dd Mg·ADP — Postrigor Open Open Down 2.1 762 1MMA 183
2 Dd Mg·ATPγS — Postrigor Open Open Down 2.1 762 1MMG 183
2 Dd Mg·AMP·PNP — Postrigor Open Open Down 2.1 762 1MMN 183
2 Dd Mg·ADP·BeF3 — Postrigor Open Open Down 2.0 762 1MMD 104
2 Dd Mg·PPi — Postrigor Open Open Down 2.7 762 1MNE 184
2 Dd Mg·ADP·BeF3 — Postrigor Open Open Down 1.75 770 1W9I 185
2 Dd Mg·ADP·BeF3 — Postrigor Open Open Down 2.0 770 1W9K 185
2 Gg Mg·ADP·AlF4 — Prepower stroke Partially closed Closed Up 3.5 820 1BR1 144
2 Gg Mg·ADP·AlF4 — Prepower stroke Partially closed Closed Up 2.9 791 1BR2 144
2 Dd Mg·ADP·VO4 — Prepower stroke Partially closed Closed Up 1.9 762 1VOM 186
2 Dd Mg·ADP·AlF4 — Prepower stroke Partially closed Closed Up 2.6 762 1MND 104
2 Dd Mg·ADP·VO4 Blebbistatin Prepower stroke Partially closed Closed Up 2.0 762 1YV3 169
2 Dd Mg·ADP·VO4 BL4 Prepower stroke Partially closed Closed Up 2.0 762 3BZ7 187
2 Dd Mg·ADP·VO4 BL6 Prepower stroke Partially closed Closed Up 2.2 762 3BZ8 187
2 Dd Mg·ADP·VO4 BL7 Prepower stroke Partially closed Closed Up 2.1 762 3BZ9 187
2 Dd Mg·ADP·AlF4 — Prepower stroke Partially closed Closed Up 2.0 770 1W9J 185
2 Dd Mg·ADP·AlF4 — Prepower stroke Partially closed Closed Up 1.95 770 1W9L 185
2 Dd Mg·ADP·BeF3 — Postrecovery stroke Partially closed Closed Up 3.6 820 1BR4 144
2 Dd Mg·ADP·VO3 — Postrecovery stroke Partially closed Closed Up 2.3 788 2JJ9 173
2 Dd Mg·ADP·VO3 PBP Postrecovery stroke Partially closed Closed Up 2.8 788 2JHR 173
2 Dd — — Rigorlike Closed Closed Down 1.9 776 2AKA/1Q5G 188
5 Gg SO4 2− — Rigorlike Closed Closed Down 2.7 766 1W8J 189
5 Gg SO4 2− — Rigorlike Closed Closed Down 2.05 795 1OE9 114
5 Gg Mg·ADP·BeF3 — Postrigor Open Open Down 2.0 795 1W7J 189
5 Gg Mg·ADP — Rigorlike Closed Closed Down 3.0 795 1W7I 189
6 Ss — — Rigorlike Closed Open Down 2.4 814 2BKH 123
6 Ss SO4 2− — Rigorlike Closed Open Down 2.9 858 2BKI 123
6 Ss Mg·ADP·BeF3 — Postrigor Open Open Down 2.4 788 2VAS 190
6 Ss Mg·ADP·BeF3 — Postrigor Open Open Down 2.3 788 2VB6 190
6 Ss Mg·ADP·VO4 — Prepower stroke Partially closed Closed Up 1.75 784 2V26 126
S1
2 Lp — — Rigorlike Closed Closed Down 2.6 839 3I5G 191
2 Lp — — Rigorlike Closed Closed Down 3.4 839 3I5H 191
2 Lp SO4 2− — Rigorlike Closed Partially closed Down 3.3 839 3I5I 191
2 Pm — — Rigorlike Closed Closed Down 3.25 838 2EC6 191
2 Lp — — Rigorlike Closed Closed Down 3.4 839 2EKV 191
2 Lp SO4 2− — Rigorlike Closed Partially closed Down 3.3 839 2EKW 191
2 Lp — — Rigorlike Closed Closed Down 2.6 839 2OVK 191
2 Pm — — Rigorlike Closed Open ? Down 3.3 840 2OS8 191
2 Gg SO4 2− — Postrigor Open Open Down ? 2.8 843 2MYS 192
2 Lp Mg·ADP — Postrigor Open Open Down 3.1 839 3I5F 191
2 Pm Mg·ADP — Postrigor Open Open Down 3.1 840 2OTG 191
2 Lp Mg·ADP — Postrigor Open Open Down 3.0 839 2OY6 191
2 Ai Mg·ADP/SO4 2− — Postrigor Open Open Down 3.1 840 1S5G 193
2 Ai SO4 2− — Postrigor Open Open Down 2.75 840 1SR6 193
2 Ai Mg2+·SO4 2− — Postrigor Open Open Down 3.2 837 1KK7 194
2 Ai — — Postrigor Open ? Down 4.2 830 1DFK 195
2 Ai Mg·ADP·VO4 — Prepower stroke Partially closed Closed Up 2.5 840 1QVI 196
2 Ai Mg·ADP·VO4 — Prepower stroke Partially closed ? Up 4.2 831 1DFL 195
2 Ai Mg·ADP — Internally uncoupled Open ? Uncoupled 2.5 835 1B7T 197
2 Ai Mg·AMP·PNP — Internally uncoupled Open ? Uncoupled 3.0 835 1KQM 194
2 Ai Mg·ATPγS p-PDM Internally uncoupled Open ? Uncoupled 3.8 835 1KWO 194
2 Ai Mg·ADP p-PDM Internally uncoupled Open ? Uncoupled 2.8 835 1L2O 194
2 Ai Mg·ADP·BeF3 — Internally uncoupled Open ? Uncoupled 2.3 837 1KK8 194

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Myosin Motors: Structural Aspects and Functionality☆

M. Preller , D.J. Manstein , in Reference Module in Life Sciences, 2017

1.3 Domain Topology and Structure of the Myosin Head

Fig. 3 shows the structure of the myosin head fragment as a ribbon diagram and a schematic topology map indicating the subdomains and their secondary structural elements. The core of the myosin motor domain is formed by a central, seven-stranded β sheet which is surrounded by α-helices. A SH3-like β-barrel domain is positioned close to the N terminus. The function of this small domain is unknown; however, it is absent in class-1 myosins and thus appears not to be essential for motor activity. A large structural domain that accounts for six of the seven strands of the central β sheet, is formed by residues 81 to 454 and 594 to 629. Unless otherwise stated, sequence numbering refers to the Dictyostelium discoideum myosin-2 heavy chain throughout this chapter. This domain is usually referred to as the upper 50-kDa domain (U50). A large cleft divides the upper 50-kDa domain from the lower 50-kDa domain (L50), a well-defined structural domain formed by residues 465 to 590. The actin binding region and nucleotide binding site of myosin are on opposite sides of the seven-stranded β sheet with the phosphate moiety of the nucleotide at the rear of the nucleotide binding pocket. The P-loop, Switch-1, and switch-2 are located in the upper 50-kDa domain close to the apex of the large cleft. All three nucleotide binding motifs contact the phosphate moiety of the nucleotide at the rear of the nucleotide binding pocket and act as γ-phosphate sensors. Structures obtained in the absence and presence of ATP analogues indicate that the switch motifs move toward each other when ATP is bound, and move away from each other in its absence. Switching between ATP and ADP states is associated with specific intramolecular movements, analogous to the nucleotide-dependent conformational transitions in G-proteins. 89 Conformational changes during the transition between different nucleotide states appear mostly to correspond to rigid-body rotations of secondary and tertiary structural elements. The motor domain can thus be regarded as consisting of communicating functional units, with substantial movement occurring in only a few residues. Residues 411 to 441 form a long helix which runs from the actin binding region at the tip of the large cleft to the fifth strand of the central β sheet. A broken helix is formed by residues 669 to 689. The broken helix is frequently referred to as the "SH1-" and "SH2-helix region," named for reactive cysteine residues. The SH1/SH2 region is tightly linked to the converter domain, which functions as a socket for the C-terminal light-chain binding domain. The relay helix, a long helix which emerges from the Switch-2 region, is in contact with the SH1 helix. As its name implies, the relay helix functions in communicating conformational information between the actin binding site, nucleotide binding site, and the mechanical amplifier elements in the neck region.

Fig. 3. (a) Ribbon representation of the chicken myosin head fragment X-ray structure (PDB2MYS) color coded for the proteolytic subdomains. Red, N-terminal SH3-like domain; blue, U50-kDa domain; green, L50-kDa domain; gray, converter domain; purple, essential light chain; pale green, regulatory light chain; yellow, light-chain binding IQ motifs. (b) Topology map of the myosin-2 motor domain. Helices are shown as cylinders and β strands as arrows.

There are approximately 100 myosin motor domain structures deposited in the protein database (see Relevant Website section) obtained by X-ray crystallography (Table 3), with most derived from Dd myosin-2. Other structures were derived from scallop (Pm and Ai), squid (Lp), chicken (Gg), or pig (Ss). Crystals were obtained under various conditions, with and without bound nucleotide or in complex with a series of nucleotide analogues. Over the last 5 years, the first crystal structures of human myosin isoforms were resolved at atomic detail, giving insights into human myosin architecture (Protein Data Bank (PDB) 2YCU, 4DB1, 4L79, 4BYF, 4P7H, 4QBD, 4PD3, 4PA0, and 4ZG4). However, there are no high-resolution structures of full-length myosins or myosins in complex with actin filaments (F-actin). Despite their highly conserved features, only the motor domains of members of four myosin classes have been characterized structurally. In addition to the various structures of class-2 myosins, the structure of three myosin-1 motor domains (PDB 1LKX, 4L79, and 4BYF), four structures of chicken myosin-5a motor domains (PDB 1W7I, 1W7J, 1W8J, and 1OE9) and one structure of human myosin-5c (PDB 4ZG4), and 20 structures of class-6 myosin motor domains (PDB 2VAS, 2VB6, 2V26, 2BKH, 2BKI, 2X51, 3L9I, 4ANJ, 4DBP, 4DBQ, 4DBR, 4E7S, 4E7Z, 4PFO, 4PFP, 4PJJ, 4PJL, 4PJM, 4PJN, 4PK4) have been solved. In other words, our understanding of structure-function relationships in the myosin and actomyosin systems is restricted to the motor domain, several more common interaction motifs in the tail region of unconventional myosins, and the neck regions of a small selection of myosin superfamily members. However, hybrid approaches, combining high-resolution structural information with results obtained using other biophysical and computational techniques, have produced insights into many aspects of the molecular mechanisms governing myosin function. 12,137 Recently solved cryoEM structures of the actin–myosin–tropomyosin complex shed new light on the interplay between the three proteins upon interaction. 138 Upon binding of the myosin motor to the actin filament, tropomyosin is shifted by 23 Å, while at the same time, the myosin motor domain is subject to a series of conformational changes involving structural elements such as the L50 domain, the central β sheet, and the N-terminal domain. Based on these structures, a model for the actin-induced myosin force generation was proposed.

Table 3. Available myosin X-ray structures in the Protein Data Bank (PDB) database (www.rcsb.org)

Myosin class Organism Ligand 1 (active site) Ligand 2 Mutation(s) State (ATPase cycle) Cleft Active site Lever arm position Resolution (Å) No. of amino acids PDB ID Ref.
Motor domain
1 Dd Mg ADP·VO4 – 20 Mut. Pre-power stroke Partially closed Closed Up 3.0 697 1LKX 90
1 (M1C) Hs Mg – – Pre-power stroke Partially closed Closed Up 2.74 725 4BYF 30
1 (M1B) Hs Mg – – Rigor-like Closed Closed Down 2.3 728 4L79 91
2 Dd Mg · ADP – 2 Mut. Post-rigor Open Open Down 2.3 776 1JWY 92
2 Dd Mg · ADP 2 Mut. Post-rigor Open Open Down 2.3 776 1JX2 92
2 Dd Mg · ADP – R238E/5 mut. Post-rigor Open Open Down 2.8 1010 1G8X 93b
2 Dd – – 2 Mut. Post-rigor Open Open Down 2.1 761 1FMV 94
2 Dd Mg · ATP – 2 Mut. Post-rigor Open Open Down 2.15 761 1FMW 94
2 Dd Mg · m-NPhAE·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 761 1D0X 95
2 Dd Mg · o-NPhAE·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 761 1D0Y 95
2 Dd Mg · p-NPhAE·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 761 1D0Z 95
2 Dd Mg·o,p-NPhAE·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 761 1D1A 95
2 Dd Mg·o,p-NPhAP·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 761 1D1B 95
2 Dd Mg·N-methyl-NPhAE·BeF3 – 3 Mut. Post-rigor Open Open Down 2.3 761 1D1C 95
2 Dd Mg · MNT – 12 Mut. Post-rigor Open Open Down 1.9 762 1LVK 96
2 Dd Mg · ADP – 3 Mut. Post-rigor Open Open Down 2.1 762 1MMA 97
2 Dd Mg · ATPγS – 11 Mut. Post-rigor Open Open Down 2.1 762 1MMG 97
2 Dd Mg · AMP·PNP – 8 Mut. Post-rigor Open Open Down 2.1 762 1MMN 97
2 Dd Mg · ADP·BeF3 – 7 Mut. Post-rigor Open Open Down 2.0 762 1MMD 98a
2 Dd Mg · PPi – 7 Mut. Post-rigor Open Open Down 2.7 762 1MNE 99
2 Dd Mg · ADP·BeF3 – S456Y/3 mut. Post-rigor Open Open Down 1.75 770 1W9I 100
2 Dd Mg · ADP·BeF3 – 3 Mut. Post-rigor Open Open Down 2.0 770 1W9K 100
2 Dd – – G680A Post-rigor Open Open Down 2.85 758 2Y0R 101
2 Dd Mg · ADP – G680A Post-rigor Open Open Down 3.13 758 2Y8I 101
2 Dd – – G680V Post-rigor Open Open Down 3.4 758 2Y9E 101
2 Dd – – S236A Post-rigor Open Open Down 2.0 762 3MYL 102
2 Gg Mg · ADP·AlF4 – – Pre-power stroke Partially closed Closed Up 3.5 820 1BR1 103
2 Gg Mg · ADP·AlF4 – – Pre-power stroke Partially closed Closed Up 2.9 791 1BR2 103
2 Dd Mg · ADP·VO4 – 7 Mut. Pre-power stroke Partially closed Closed Up 1.9 762 1VOM 104
2 Dd Mg · ADP·AlF4 – 7 Mut. Pre-power stroke Partially closed Closed Up 2.6 762 1MND 98a
2 Dd Mg · ADP·VO4 Blebbistatin 6 Mut. Pre-power stroke Partially closed Closed Up 2.0 762 1YV3 105
2 Dd Mg · ADP·VO4 BL4 – Pre-power stroke Partially closed Closed Up 2.0 762 3BZ7 106
2 Dd Mg · ADP·VO4 BL6 – Pre-power stroke Partially closed Closed Up 2.2 762 3BZ8 106
2 Dd Mg · ADP·VO4 BL7 – Pre-power stroke Partially closed Closed Up 2.1 762 3BZ9 106
2 Dd Mg · ADP·AlF4 – S456Y/2 mut. Pre-power stroke Partially closed Closed Up 2.0 770 1W9J 100
2 Dd Mg · ADP·AlF4 – S456E/3 mut. Pre-power stroke Partially closed Closed Up 1.95 770 1W9L 100
2 Dd Mg · ADP·VO4 27X – Pre-power stroke Partially closed Closed Up 2.5 776 4AE3 107
2 Dd Mg · ADP·VO4 – S456Y Pre-power stroke Partially closed Closed Up 2.4 692 3MKD 108
2 Dd Mg · ADP·VO4 Blebbistatin S236A Pre-power stroke Partially closed Closed Up 2.01 762 3MYH 102
2 Dd Mg · AMP·PNP Blebbistatin S236A Pre-power stroke Partially closed Closed Up 1.84 762 3MYK 102
2 Gg Mg · ADP·BeF3 – – Post-recovery stroke Partially closed Closed Up 3.62 820 1BR4 103
2 Dd Mg · ADP·VO3 – – Post-recovery stroke Partially closed Closed Up 2.3 788 2JJ9 109
2 Dd Mg · ADP·VO3 PBP – Post-recovery stroke Partially closed Closed Up 2.8 788 2JHR 109
2 Dd Mg · ADP·VO3 PClP – Post-recovery stroke Partially closed Closed Up 2.5 776 2XEL 110
2 Dd Mg · ADP·VO3 TBDClP – Post-recovery stroke Partially closed Closed Up 2.4 776 2XO8 111
2 Dd Mg · ADP·VO3 KI9 3 Mut. Post-recovery stroke Partially closed Closed Up 2.7 695 2X9H 112
2 Dd Mg · ADP·VO3 Blebbistatin – Post-recovery stroke Partially closed Closed Up 2.2 788 3MJX 109
2 Dd Mg · ADP·VO3 Resveratrol – Post-recovery stroke Partially closed Closed Up 2.2 788 3MNQ 113
2 Dd Mg · ADP·PO4 – R238E/E459R Pi release Partially closed Open Up 2.15 769 4PJK 114
2 Dd – – – Rigor-like Closed Closed Down 1.9 776 2AKA 115
2 (β-card) Hs Mn · AMP·PNP – – Post-rigor Open Open Down 2.6 783 4DB1 116
2 (β-card) Hs SO4 2− – – Post-rigor Open Open Down 3.2 1025 4P7H 117
2 (β-card) Hs – Omecamtiv mercarbil – Post-rigor Open Open Down 2.25 1026 4PA0 117
2 (NM2C) Hs Mg · ADP·VO4 – – Pre-power stroke Partially closed Closed Up 2.25 995 5I4E/2YCU 118
2 (NM2B) Hs – – – Rigor-like Closed Closed Down 2.84 1032 4PD3 119
5 Gg Mg · ADP·BeF3 – – Post-rigor Open Open Down 2.0 795 1W7J 120
5 Gg SO4 2− – – Rigor-like Closed Closed Down 2.7 766 1W8J 120
5 Gg SO4 2− – – Rigor-like Closed Closed Down 2.05 795 1OE9 121
5 Gg ADP – – Rigor-like Closed Closed Down 3.0 795 1W7I 120
5 (M5C) Hs Mg · ADP·VO4 – – Pre-power stroke Partially closed Closed Up 2.36 764 4ZG4 122
6 Ss Mg · ADP·BeF3 – 6 Mut. Post-rigor Open Open Down 2.4 788 2VAS 123
6 Ss Mg · ADP·BeF3 – 10 Mut. Post-rigor Open Open Down 2.3 788 2VB6 123
6 Ss Mg · ADP·BeF3 – D179Y Post-rigor Open Open Down 2.6 788 4DBQ 124
6 Ss Mg · ADP PO4 3− R393M Post-rigor Open Open Down 2.4 788 4PJJ 114
6 Ss Mg · ADP·VO4 – 6 Mut. Pre-power stroke Partially closed Closed Up 1,75 784 2V26 125
6 Ss Mg · ADP·VO4 – – Pre-power stroke Partially closed Closed Up 2.3 798 4E7Z 126
6 Ss Mg · ADP·VO4 PO4 3− 3 Mut. Pre-power stroke Partially closed Closed Up 2.25 798 4E7S 126
6 Ss Mg · ADP·AlF4 – 11 Mut. Pre-power stroke Partially closed Open Up 2.6 817 4ANJ 126
6 Ss Mg · ADP·VO4 – D179Y Pre-power stroke Partially closed Open Up 1.95 786 4DBR 124
6 Ss Mg · ADP·PO4 – – Pre-power stroke Partially closed Closed Up 2.78 788 4PK4 114
6 Ss Mg · ADP – – Pi release Partially closed Open Up 1.75 788 4PFO 114
6 Ss Mg · ADP·PO4 – – Pi release Partially closed Open Up 2.32 788 4PFP 114
6 Ss Mg · ADP – A458E Pi release Partially closed Open Up 2.1 788 4PJL 114
6 Ss Mg · ADP·PO4 – – Pi release Partially closed Open Up 2.05 788 4PJM 114
6 Ss Mg · ADP PO4 3− – Pi release Partially closed Open Up 2.0 788 4PJN 114
6 Ss – – 6 Mut. Rigor-like Closed Open Down 2.4 814 2BKH 127
6 Ss SO4 2− – – Rigor-like Closed Open Down 2.9 858 2BKI 127
6 Ss SO4 2− – 7 Mut. Rigor-like Closed Open Down 2.2 789 2X51 128
6 Ss – – L310G/8 mut. Rigor-like Closed Open Down 2.2 816 3L9I 129
6 Ss – – D179Y Rigor-like Closed Open Down 2.2 814 4DBP 124
S1
2 Lp – – E238K/2 mut. Rigor-like Closed Closed Down 2.6 839 3I5G 130
2 Lp – – E238K/2 mut. Rigor-like Closed Closed Down 3.4 839 3I5H 130
2 Lp SO4 2− – E238K/2 mut. Rigor-like Closed Partially closed Down 3.3 839 3I5I 130
2 Pm – – 7 Mut. Rigor-like Closed Closed Down 3.25 838 2EC6 130
2 Pm – – – Rigor-like Closed Open? Down 3.3 840 2OS8 130
2 Gg SO4 2− – 74 Mut. Post-rigor Open Open Down? 2.8 843 2MYS 244
2 Lp Mg · ADP – E238K/2 mut. Post-rigor Open Open Down 3.1 839 3I5F 130
2 Pm Mg · ADP – – Post-rigor Open Open Down 3.1 840 2OTG 130
2 Ai Mg · ADP/SO4 2− – – Post-rigor Open Open Down 3.1 840 1S5G 131
2 Ai SO4 2− – – POST-rigor Open Open Down 2.75 840 1SR6 131
2 Ai Mg2+ · SO4 2− – – Post-rigor Open Open Down 3.2 837 1KK7 132
2 Ai − – – Post-rigor Open ? Down 4.2 830 1DFK 133
2 Ai Mg · ADP·VO4 – – Pre-power stroke Partially closed Closed Up 2.5 840 1QVI 134
2 Ai Mg · ADP·VO4 – – Pre-power stroke Partially closed ? Up 4.2 831 1DFL 133
2 Ai Mg · ADP − – Internally uncoupled Open ? Uncoupled 2.5 835 1B7T 135
2 Ai Mg · AMP·PNP – – Internally uncoupled Open ? Uncoupled 3.0 835 1KQM 132
2 Ai Mg · ATPγS p-PDM – Internally uncoupled Open ? Uncoupled 3.8 835 1KWO 132
2 Ai Mg · ADP p-PDM – Internally uncoupled Open ? Uncoupled 2.8 835 1L2O 132
2 Ai Mg · ADP·BeF3 – – Internally uncoupled Open ? Uncoupled 2.3 837 1KK8 132
2 Dm – – – Rigor-like Closed Open Down 2.23 818 4QBD 136

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Muscle and Nonmuscle Contractile Systems

N.V. BHAGAVAN , in Medical Biochemistry (Fourth Edition), 2002

Mechanism of Contraction: Cross-Bridge Cycling

The nature of the S2 region allows the myosin heads to be in very close proximity to the actin. When the tropomyosin-mediated inhibition of contraction is reversed, the myosin heads interact strongly with actin. Although there is still debate over the details of how this interaction produces mechanical work, there is a consensus on the general outline. Myosin bonds initially via relatively flexible loop regions near the catalytic site, followed by progressively more extensive hydrogen bonding. In this process, a large surface area on each protein (totaling 15-20 nrn 2) is removed from interaction with cell water during formation of the bond between them. In cases where proteins bond without changes in conformation, burying this much area would be associated with an energy change of 60-80 kJ/mol of bonds, a number roughly twice as great as that actually observed for binding of myosin to actin. This suggests the capture of energy internally in the myosin head, actin, or both. Apparently, as the myosin moves into this tightly bound configuration, energy is captured in the form of deformation within the myosin head, which applies force (5-10 piconewtons) to the head's attachment to the thick filament (the neck region). Much, probably most, of this energy is available to do mechanical work as the filaments slide past one another (the power stroke illustrated schematically in Figure 21-10), with myosin ending up very firmly bound to actin. Subsequent binding of Mg-ATP to myosin, which is also a very high-affinity binding, provides the free-energy input to the system to alter myosin's actin binding site to a lowaffinity state, permitting detachment. ATP binding lowers the affinity of myosin for actin by a factor of about 104. Hydrolysis of the ATP then occurs, with little change in the free energy of the system. Subsequent events release Pi and ADP from the ATPase site.

FIGURE 21-10. Schematic illustration of the sliding filament-rotating head mechanism of force generation in muscle. Cross-bridges form approximately at right angles to the thin filaments (a). This angle changes to about 45 degrees at the end of the cross-bridge cycle when the bridge is released. Recent measurements indicate that the initial and final angles in intact sliding filaments are more nearly 80 and 50°, respectively. In the attached bridge, a conformational change occurs, putting tension on the neck region. This may be due to an abrupt change in the angle of the head (b). Movement of the thick and thin filaments relative to each other relieves the stress on the neck. The product of the tension (force) and the distance moved is the work done per stroke by a cross-bridge.

Thus, in the cross-bridge cycle, myosin is bound with high affinity alternately to actin and to ATP. Since the energy changes associated with myosin binding to actin and MgATP are internal to the system, the only free energy changes externally observable are the free-energy change from ATP in solution to ADP and Pi in solution, which equals the sum of the mechanical work performed plus the heat released. Thus the overall result is the conversion of energy of hydrolysis of ATP (about 50 kJ/mol under physiological conditions) to work (and heat), a process called chemomechanical transduction. The efficiency of this process in mammalian skeletal muscle is 60-70%.

There is uncertainty over the timing of ATP hydrolysis and release of ADP and Pi with respect to the actomyosin binding states and the power stroke. It is currently thought that ATP hydrolysis occurs after transition of the actomyosin-ATP (A-M-ATP) complex to a weakly-bound state and may sometimes occur after release of myosin from actin. The complex A-M-ADP-Pi may remain weakly bound until dislodged by movement of the filaments. The released M-ADP-Pi has moderate affinity for actin, and upon reattachment, forming A-M-ADP-Pi, the phosphate release step occurs. This creates a state called A-M*-ADP which is the high-affinity state associated with initiating the power stroke. As the structural changes produced by increasingly tight binding to actin produce strain in the myosin head and therefor force and movement, the affinity of the ATPase site for ADP also changes, releasing the ADP. Thus, at the end of the power stroke, cross-bridges are typically in the A-M state (called the rigor state), their most tightly bound rigid state, in which they will remain unless ATP is available to bind to the ATPase site and alter the affinity of the actin binding site (Figure 21-11). In normal circumstances it is almost impossible to deplete ATP to the point that a large proportion of myosin heads form rigor bonds, but it does happen in severely ischemic muscle and post-mortem (rigor mortis). When Mg-ATP is available, binding occurs and alters the actin binding site to a low-affinity configuration, and hydrolysis follows, so that the cross-bridge will probably be in the weakly-bound A-M-ADP-Pi state until once again pulled free. So long as [Ca2+]i remains high, this cycle will continue, provided that adequate ATP concentration and other appropriate conditions of the internal environment are maintained. The rate-limiting step is the Pi release step, and all of the steps following Pi release up through ATP hydrolysis happen quickly, so that the M-ADP-Pi and A-M-ADP-Pi states predominate.

FIGURE 21-11. A simplified model of the interactions thought to occur during the cross-bridge cycle. Four major structural states are depicted, but the actual number of structurally and energetically distinct states through which this system transitions is uncertain. The vertical drop in the figure crudely represents the free-energy change in the transitions shown. The functional change in myosin structure is shown as a change in the relative orientation of the neck (cross-hatched) to the catalytic head region (hatched). A is actin, M is myosin, and Pi is phosphate. M. ATP represents myosin complexed with ATP immediately after detachment from actin. Here, the neck is shown in the same configuration as at the end of the power stroke. Hydrolysis of ATP leads to M. ADP. Pi, with weak bonding to actin both before and after hydrolysis. ATP hydrolysis is energetically favored, but due to the products remaining bound, the free-energy change is not large. Hydrolysis is shown here as eliciting a change in orientation of the neck, producing the conformation thought to exist at the initiation of the power stroke, but the relationship between the hydrolysis events and the mechanical events is still speculative. Release of Pi from A. M. ADP. Pi following binding to actin is associated with conformational changes that produce the strained, or force-producing, state A. M* · ADP. Transition from A. M* · ADP to A · M coincides with performance of mechanical work and formation of a very strong A · M bond, and a reorientation of the neck region is thought to occur during this transition. In striated muscle, a large part of the free energy driving the cross-bridge cycle is lost between these two states. A · M · ADP and A · M are thought to differ little in configuration, and the energy change associated with ADP release is small. Subsequent binding of ATP to A. M dramatically lowers the affinity of myosin for actin, probably producing release of M. ATP from A without altering the neck orientation, returning the system to the state shown at the upper left. [Adapted from: R. Cooke, Actomyosin interaction in striated muscle. Physiol. Rev. 77(3): 671-697, 1997.]

In a general way, transport ATPases are similar to myosin. The initial binding of the transported substance to the transport protein corresponds to tight binding of myosin to actin. Reorientation of the binding site toward the opposite face of the membrane is analogous to the force-producing conformational change in the myosin head, and conversion of the substrate binding site to a low-affinity state is accomplished by binding of ATP. ATP hydrolysis is associated with return of the substrate binding site to its original orientation, and ADP and Pi are released into solution.

It is important to note that this model predicts the hydrolysis of one ATP for every cross-bridge cycle of every myosin head: there is evidence that, in high-speed contractions at least, there may be multiple attachments and detachments per hydrolysis. Thus, our understanding is obviously not complete. Nevertheless, the ATP consumption associated with contraction can be enormous. The metabolic scope (ratio of maximal to resting energy consumption) of skeletal muscle can reach 100:1, and there must necessarily be metabolic specializations to meet this peak demand and to do so quickly. Energy metabolism is discussed in Chapters 13–15 13 14 15 and 18.

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Clinical Significance of Cardiac Contractile Proteins for the Diagnosis of Myocardial Injury

Johannes Mair , ... Gerd Michel , in Advances in Clinical Chemistry, 1994

1.2.1.1 MLCs

The four MLCs are associated with the two myosin heads. The bound light chains consist of a pair of regulatory MLCs (MLC-2) and a pair of alkali (essential) MLCs (MLC-1). One MLC-1 and one MLC-2 subunit are associated with the globular head region of each MHC (see Fig. 3). Although the role of the light chains is poorly understood, their location near the hinge region suggests that they may be involved in modulating interactions between myosin and actin (H2, W3). The slow skeletal muscle MLC-1 and MLC-2 appear to be identical with the cardial MLC-1 and MLC-2 (P2). In mice and rats both slow skeletal MLC-1 and cardiac MLC-1 are encoded by a single gene (Bl). Amino acid sequence analyses strongly indicate the identity of slow skeletal MLC-2 and cardiac MLC-2 in rabbits and chickens. A minor fraction of MLC (~1% of the total MLC content) exists as a soluble cytoplasmic precursor pool for myosin synthesis (H6, K12). The cytosolic pool of TnT is ~50 times larger compared with MLC-1 (K10).

Myofibrillar proteins of striated muscles are expressed as tissue-specific isoforms and consequently these antigens may be differentiated by immunological methods. However, myosin gene regulation is complex. The degree to which cardiac slow and fast skeletal muscle genes are coexpressed or have "overlapping" expression is striking. For example, cardiac MLC, cardiac β-type MHC, cardiac tropomyosin, and cardiac TnC are coexpressed in slow skeletal muscle. By contrast, currently it appears that cardiac TnT and TnI are molecules unique to human myocardium in adults, where they are the only TnT and TnI isotypes present in normal and diseased myocardium. Thus, cardiac TnT and TnI have the best potential of all human cardiac contractile proteins for the development of cardiac-specific immunoassays. A survey of the literature demonstrates that all currently described MHC, MLC, and tropomyosin assays showed varying but significant cross-reactivity with their isoforms isolated from skeletal muscle (C6, G1, K2, K7, L2, L5, M12, S7, W2, Y2). The superiority of these assays to CKMB or LDH-1 with respect to diagnostic specificity has not yet been clearly demonstrated. By contrast, cardiac-specific TnI and TnT immunoassays have been described without cross-reactivity with their skeletal muscle isoforms (C3, C4, K8, L3).

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Cardiac Energetics

June-Chiew Han , ... Denis S. Loiselle , in Muscle and Exercise Physiology, 2019

23.5.2 Ca2+ Activation

We define a cross-bridge as the interaction of a myosin head with a binding site on actin. In the absence of Ca 2+, access to the binding site on actin is blocked, preventing myosin from binding. Here the cross-bridges are in a nonpermissive state, N XB (Fig. 23.15). When intracellular Ca2+ rises as a result of the Ca2+-induced-Ca2+-release (CICR) process, the binding of Ca2+ to tropomyosin removes the allosteric inhibition and permits the binding of myosin to the actin site. This places the cross-bridges in the permissive state (P XB) where they are free to cycle. Ca2+ activation is a highly nonlinear process and the steep sensitivity of force as a function of Ca2+ arises from the nearest-neighbor interactions of troponin and tropomyosin along the thin filament. In the model, this is captured using a phenomenological representation of the nearest-neighbor interaction and is described by the transition rates K npT and K pnT. These two parameters describe the rate at which the cross-bridge transitions between N XB and P XB, respectively. An increase in intracellular Ca2+ concentration increases the value of K npT, shifting the cross-bridge to the permissive state. Note that K npT is also a function of H+, allowing the model to capture the competitive binding of H+ to the Ca2+-binding site on troponin (Tran et al., 2010).

Figure 23.15. Schematic of the cross-bridge cycle.

(A) The full cycle. (B) The simplified model, in which the two strongly post-rotated states, AM1 and AM2, are collapsed into a single state, XB postR, by assuming rapid equilibrium of MgADP binding.

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Striated Muscle Dynamics

S.K. Gollapudi , ... M. Chandra , in Reference Module in Biomedical Sciences, 2014

Thick Filaments

The main component of the thick filament is myosin-II (two myosin heads); each myosin-II consists of two identical myosin heavy chains (MHC). Each MHC consists of a globular head region connected to a long coiled-coil tail region via an elastic hinge region ( Figure 4). Coiled-coil tail regions of myosin-II polymerize to form the thick filament, while the globular head regions protrude from the thick filament at regular intervals. These globular myosin heads can interact with actin to form crossbridges during muscle activation. Myosin heads are oriented toward the Z-line, while the coiled-coil tails are directed toward the M-line. An important feature of myosin polymerization is that myosin-II molecules are oriented in opposite directions in each half of the sarcomere. This orientation results in polarized thick filaments that help in directing force toward the center of the sarcomere. Each thick filament is made up of ∼300 myosin-II molecules. Each globular head of myosin contains an ATPase site and an actin-binding site. The energy released from the hydrolysis of adenosine triphosphate (ATP) and the structural rotation of myosin heads, with respect to the thin filament, produce force and shortening.

Figure 4. Structural organization of thick and thin filament proteins.

 Illustrated pictures are reproduced from Martini, F., 2006. Fundamentals of Anatomy and Physiology, seventh ed. Pearson Education Inc., San Francisco, CA, p. 291, with permission.

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