The polymerization of monomeric G-actin into F-actin and the reverse depolymerization of F-actin into G-actin monomers are dynamic processes, employed by eukaryotic cells in a plethora of biochemical contexts (e.g. cytokinesis, cell motility, cytoplasmic movement, vesicle transport, sperm acrosomal reactions, and the maintenance of cell morphology). In animals, F-actin is a key player in the transduction of chemical energy into mechanical energy during muscle contraction.
G-actin is a globular protein of ~43-kDa that binds an adenine nucleotide, stabilizing the monomer. Binding of ATP usually precedes polymerization into F-actin microfilaments and ATP---> ADP hydrolysis normally occurs after filament formation such that newly formed portions of the filament (with bound ATP) can be distinguished from older portions (with bound ADP). This may play a role in microfilament dynamics.
This tutorial will examine G-actin and F-actin structures from several sources, concluding with a focus on the interaction of F-actin with myosin and other proteins in the sarcomeres of animal myofibrils.
II. Actin Structures
To the left is a G-actin monomer from the yeast, Saccharomyces cerevisiae. The protein binds ATP in a deep cleft formed by two major domains, 1 and 2.
Domain 1 is subdivided into subdomains 1 and 2, and domain 2 is subdivided into subdomains 3 and 4.
At the bottom of cleft are two residues that are important to ATP hydrolysis and therefore to microfilament dynamics. Peering into the cleft, it can be seen that histidine 161 (from subdomain 3) and glutamine 137 (from subdomain 1) are involved in positioning a key water oxygen (blinking) for a nucleophilic attack of the gamma phosphate (blinking) of ATP. A magnesium ion is involved in stabilizing the terminal phosphates of ATP as catalysis proceeds. Note that the nucleotide contacts all 4 subdomains. ATP hydrolysis can thus change the conformation of the actin monomer.
Particularly plastic is a loop in subdomain 2 of actin with bound ATP (PDB ID 1YAG). This is observed to fold into an α-helix in the ADP-bound form of G-actin (from rabbit, PDB ID 1J6Z). The loop/helix plays a role in intermonomer interactions in polymeric F-actin (see below), so ATP hydrolysis can impact the stability of actin filaments.
A simulation of the folding changes induced by ATP hydrolysis is shown at left. Although the starting and ending structures are genuine models based on crystallographic data (PDB ID's 1YAG and 1J6Z), the intermediate transitions are based on linear interpolation and some energy minimization and are only possible structures. As above, note the folding of the loop in subdomain 2 into an α-helix as ATP (not shown) is hydrolyzed. This simulation was generated using the Yale Morph Server at the Database of Macromolecular Movements, maintained by the Gerstein lab. Note: if you are prompted, choose continue script.
A model of F-actin has been produced by fitting different crystal structures of G-actin (both ATP and ADP-bound forms) into an atomic density map derived from cryo-electronmicroscopic examination of F-actin bundles from the acrosomal reaction of horseshoe crab (Limulus) sperm (Cong, et al., 2008). The microfilament consists of a helical arrangement of two actin strands, each formed by polymerization of G-actin monomers. The twist of the helix can be influenced by the cellular environment, including actin binding proteins.
III. Actin/Tropomyosin/Troponin in the Thin Filaments of the Sarcomere
Actin filaments are key components of the sarcomeres, the fundamental units of muscle contraction. At this point it is recommended that you consult a textbook to review the molecular architecture of muscle myofibrils and sarcomeres and the mechanism of calcium release from intracellular stores in muscle cells in response to neuronal stimulation. Also, see Figure 1 and Figure 2.
Thin filaments in the sarcomere, comprising F-actin, tropomyosin, and troponin proteins, are the substrate upon which the myosin molecules of the thick filaments exert their pull, thus transducing the chemical energy of ATP hydrolysis into a mechanical force that contracts individual myofibrils within muscle cells.
A length of F-actin in a thin filament is shown at left. The orientation of the helical filament is such that the Z-line is to the right and the M-line is to the left in the sarcomere.
Two tropomyosin molecules are wound around the actin polymer. Each consists of a coiled-coil, i.e. two intertwined α-helices. Each helix contacts several actin monomers in the filament.
Troponin protein complexes are associated with each tropomyosin molecule, separated by the approximate length of of tropomyosin, ~39 nm (390 Å).
Each troponin complex is a heterotrimer that contains one troponin C, one troponin T, and one troponin I component. Note the close apposition of an arm of troponin T with tropomyosin.
Each troponin C from skeletal muscle can bind 4 calcium ions via 4 EF-hand domains. These are highly conserved structural motifs consisting of Ca++-binding loops flanked by alpha-helices found in other Ca++-binding proteins, e.g. calmodulin
We can now consider the structural regulation of thin filament structure in skeletal muscle in response to an action potential. When a postsynaptic muscle cell receives neurotransmitter from the presynaptic neuron, rapid depolarization of the muscle cell membrane due to Na+ influx is propagated to the sarcoplasmic reticulum via T tubules. This results in the opening of Ca++ channels and the flow of Ca++ ions into the myoplasm from the intracellular stores in the reticulum.
Ca++ then binds to troponin C in the troponin complex, which ultimately causes a shift in position of tropomyosin molecules on the actin filament. This exposes sites on actin monomers in the filament that can be bound by the molecular motor, myosin, the major protein of the thick filaments within the sarcomere. See Figure 1 and Figure 2.
Myosin conformational shifts driven by ATP ---> ADP hydrolysis, release of Pi and ADP, and re-binding of ATP cause rapid cycles of actin binding and release, driving contraction of muscles (see section IV, below).
IV. Myosin II Structural Conformers and the Actomyosin Cycle
Myosin II is the motor protein of the sarcomeric thick filament that transduces the chemical energy of ATP hydrolysis and release of ADP and Pi into mechanical energy that drastically alters the conformation of the protein. This allows myosin to undergo repeated cycles of actin binding and release (the actomyosin cycle). The conformational movements of portions of myosin are amplified to produce the mechanical force that drives the sliding of thin filaments, thus shortening the sarcomere and causing muscle contraction. Myosin II structure and its interaction with actin will be considered in the remaining part of this tutorial. Before embarking on the role of myosin II conformational changes in the actomyosin cycle, it is highly recommended that you familiarize yourself with this cycle as described in a texbook. Briefly summarized, the cycle involves 4 steps:
Each myosin molecule in the thick filament is composed of two myosin heavy chains and two pairs of light chains (for reference, see Figure 2). At left is displayed only the distal portion of one myosin heavy chain (head = motor domain and neck = lever arm) with two light chains bound to the lever arm. The heavy chain motor domain plus lever arm is referred to as subfragment 1 (S1) of myosin II. The S1 subfragment shown is from myosin II of striated muscle of a vertebrate (chicken). The extended tail of the myosin molecule, which forms a coiled-coil with another myosin II heavy chain, is not shown.
The myosin II light chains, are tandemly bound to the heavy chain lever arm. The regulatory light chain and the essential light chain serve regulatory roles and can also stabilize the lever as well as provide for association of dimerized heavy chains with other myosin II molecules in the thick filament. This is promoted by phosphorylation of the light chains by myosin light chain kinase (MLCK) or Rho-associated kinases. Both light chains show structural homology with calmodulin.
Turning now to the structure of the heavy chain motor domain, four major subdomains plus several elements that articulate movements between the subdomains and between the motor domain and the lever have been described.
The subdomains are the lower subdomain (LS) , the upper subdomain (US), the SH3 motif (SH3), and the N-terminal subdomain (NT). The articulation structures are the converter, the relay, switch II, and the SH1 helix.
Changes in the relative dispositions of the major subdomains of the motor domain (LS, US, SH3, NT) are effected by ATP binding, ATP hydrolysis, release of Pi, and release of ADP (see the actomyosin cycle steps). These biochemical functions affect the ability of myosin to bind actin and alter the strength of actin binding. Also, the shifts of the subdomains are amplified by the relay and converter, such that large swings of the lever relative to the motor domain occur. The motions of these various elements as the actomyosin cycle progresses will be seen to be important, so it is suggested that you familiarize yourself with their relative positions before proceeding.
Before examining the structural changes in myosin with the steps of actomyosin cycle, let's identify the the actin binding cleft and the nucleotide binding cavity (catalytic site) in the motor domain.
We now can address the correlation between some of the myosin S1 conformers and particular transitions within the actomyosin cycle. In the following, a black line indicates the approximate direction of the actin thin filament axis relative to the motor domains of S1. The following morphed simulations of conformational shifts were generated using the Yale Morph Server at the Database of Macromolecular Movements, maintained by the Gerstein lab. As for the actin morphs, above, the starting and ending structures are genuine models based on crystallographic data (PDB ID's 1B7T, 1DFL, 1DFK), but the intermediate transitions are based on linear interpolation and some energy minimization and are only possible structures. The S1 structures are all from scallop muscle myosin II and are hypothesized to closely represent the state of the myosin S1's at the indicated steps of the cycle. Bound nucleotides and Pi's are not represented. In order to observe details of the simulated intramolecular motions, it may be necessary to repeat each step multiple times.
First, consider the pre-powerstroke ---> post-powerstroke transition (see steps 1-2, outlined above). Observe the profound shifting of the lever arm relative to the motor domain. In the pre-powerstroke configuration, the lever arm is nearly perpendicular to the thin filament axis, whereas it lies at a nearly 45o angle relative to this axis at the end of the powerstroke. Also quite noteworthy are the movements of the relay, the SH1 helix, and especially the converter as these elements amplify the relatively small shifts in the primary motor subdomains (LS, US, SH3, NT) in response to ADP and Pi release, thus driving the large lever arm motions. The relay has strong connections to the converter and is thought to control its movements. The relay, the SH1 helix, and the converter have been analogized to components of a "transmission" that connects the motor subdomains to the lever.
Remember that S1 lever arms are continuous with the myosin II tails in intact myosin molecules within the sarcomeric thick filaments (see Figure 1). The thick filaments, and the tails of myosin II, run in parallel to the the thin filaments. Given this, It should be possible to envision how the powerstroke slides the thin filament past the thick filaments, as the lever arms rotate relative to both the thick and thin filament axes.
The post-powerstroke ---> release-of-actin transition (see steps 2-3, outlined above) is driven by the binding of ATP (not shown) in the nucleotide binding pocket of the S1 motor domain. Again, this transition involves considerable shifts in the relative orientation of the lever arm and motor domain.
A distinctive closing of the actin binding cleft between the LS and US in this step accounts for the release of thin filament actin.
The step 2--->3 conformational change of the SH1 helix is of interest. It can be seen to unwind as this transition takes place. In a "clutch-like" role, it is thought that this disordering effectively uncouples the converter and lever arm from the motor, with important functional consequences. It is possible that a myosin-ATP <---> nucleotide-free-myosin equilibrium exists at this step. If the SH1 helix did not uncouple the lever arm from the motor, immediate reattachment of the myosin to actin might occur. This would block filament sliding. Also, the unwinding of the SH1 helix increases mechanical freedom for the converter and lever arm. This could ready myosin for the transition to the pre-powerstroke conformation.
Finally, hydrolysis of ATP produces a transition state intermediate with bound ADP and Pi in the active site, cocking the myosin head in preparation for for weak binding to actin.
We can conclude this section by stating the obvious: the tremendous, dynamic conformational plasticity of myosin II is an important and amazing aspect of its function.
V. Myosin - Actin Interaction
The interaction of a myosin II S1 subfragment with an actin filament has been modeled. As can be observed, actin binding is mediated by residues in the upper and lower subdomain cleft. Residues 335-372 in an actin monomer of the filament show the most extensive contact with these loops. This actin site has been shown to be covered by tropomyosin in relaxed vertebrate thin filaments, accounting for the steric blocking of myosin binding by tropomyosin.
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