Figure 1.
The Lymn–Taylor functional cycle of the actomyosin complex [6],[13] (adapted from Yu et al. [19] to indicate the motion of the lever arm appropriate for myosin V).
Only a myosin monomer is shown for simplicity. Binding of ATP to the actomyosin complex (the rigor state) leads to rapid dissociation of myosin from actin without immediate hydrolysis of ATP. Coupled with a major structural change in the orientation of the lever arm (“recovery stroke”), ATP hydrolysis proceeds and the motor domain weakly rebinds to actin. Following the release of Pi, the motor domain undergoes the “powerstroke” during which the lever arm moves back to the rigor state and the motor domain becomes strongly bound to actin. Dissociation of ADP leads the system back to the rigor state.
Figure 2.
The myosin motor domain presented in the rigor-like conformation.
(A) The motor subdomains and its functional sites. The nearly rigid motor subdomains are shown in space-filling models (on the left) and cartoons (on the right). The N-terminal (N), the upper 50 kDa (U50), the lower 50 kDa (L50), the converter (C), the first IQ motif (IQ), and the essential light chain (ELC) are colored in orange, blue, red, lime, pale green, and yellow, respectively. In the space-filling representation the location of the myosin functional sites is indicated: the actin-binding site at the interface of the U50 and L50 subdomains; the nucleotide-binding site at the interface of the N and U50 subdomains; and the beginning of the lever arm, whose position is controlled by the rotation of the converter. (B) The subdomain connectors in the myosin motor domain. The various connectors are color-coded as follows: the P-loop, switch I, switch II, the strut, the relay, helix SH1, and loop 76–81 are cyan, magenta, orange, red, yellow, slate, and violet. The P-loop, switch I and switch II contribute to the formation of the active site involved in nucleotide binding and hydrolysis at the interface of the N, U50, and L50 subdomains; the strut joins U50 and L50 in the upper part of the U50/L50 cleft; the relay group connects L50 to C; helix SH1 and loop 76–81 connect the converter to the N-terminal subdomain. In the text, the terms “upward” and “downward” are used to indicate motion in the direction of the top and bottom of the figure.
Figure 3.
Rigor-like/post-rigor normal mode overlaps (see text).
Dark colors indicate large overlaps (values close to unity) and correspond to strongly correlated motions.
Figure 4.
Involvement-coefficient analysis of the rigor-like/post-rigor conformational transition (see text).
Individual (A) and cumulative (B) involvement coefficients are shown for the “forward” (from rigor-like to post-rigor) and “backward” (from post-rigor to rigor-like) transitions in red and green, respectively. Negative indices correspond to pure translational and rotational modes.
Figure 5.
Involvement coefficients specialized for the rigor-like/post-rigor transition of the head domain and the neck region.
The frequency range for the specialized modes is indicated.
Table 1.
Rigor-like and post-rigor involvement coefficients for the conformational transition of the entire molecule (head plus neck), the head domain (N, U50, and L50; aa 61–699) and the neck region (C, IQ, and ELC; aa 700–946).
Table 2.
Subdomain coupling on the most-involved low-frequency modes.
Figure 6.
Rigor-like and post-rigor lowest-frequency modes that are essentially independent of the motor head.
(A) Amplitude of the Cα fluctuations along the sequence computed upon optimal superposition of the head domain (aa 61–699) for the three lowest-frequency modes. Black and red profiles correspond to the rigor-like and post-rigor states, respectively. In the background, pink, light blue, grey, light green, cyan, and yellow indicate the boundaries of the N, U50, L50, C, IQ, and ELC subdomains. Rigor-like and post-rigor fluctuations show a difference in the conformational freedom of the converter in the two states (light green region). (B) DynDom analysis of the rigor-like and post-rigor lowest-frequency modes. In both cases two dynamic domains corresponding to the head domain (red) and the neck region (blue) are identified. The analysis indicates that the converter subdomain (shown surrounded by a dashed circle) belongs to the head domain in the rigor-like state and to the neck region in the post-rigor state.
Figure 7.
Lever-arm motion encoded in the lowest-frequency modes in the rigor-like and post-rigor states of myosin V.
(A) Pictorial representation of the lever oscillation along modes 1 (red) and 3 (blue) in the rigor-like and post-rigor states. (B) Lever-arm motion reported in spherical coordinates along the lowest-frequency modes. The spherical coordinates φ and θ, which correspond to the zenith and the azimuth angle, respectively, were determined by fitting the coordinates of the lever-arm backbone atoms (aa 754–792) upon optimal superposition of the N, U50, and L50 subdomains to the equilibrium structure. The circle and square correspond to the orientation of the lever arm in the rigor-like and post-rigor conformations, respectively. Mode 1 and 3 are essentially perpendicular in both functional states.
Figure 8.
Cα-RMS deviation of the NMSM post-rigor conformation from the “target” (i.e., the X-ray post-rigor conformation after energy minimization) as a function of the number of low-frequency rigor modes included in the optimal superposition.
The rigor modes were first sorted according to their rigor-like/post-rigor involvement coefficients and then combined as described in Materials and Methods. Only 14 modes are sufficient to obtain a NMSM conformation that is at 1.5 Å RMSD from the target structure. The mode indexes of these 14 highly involved modes are indicated.
Table 3.
Rigid-body description of the NMSM transition in terms of subdomain screw-axis transformations.
Figure 9.
The rigor-like to NMSM post-rigor conformational transition.
The energy-minimized rigor structure is color-coded as in Figure 2. The NMSM post-rigor conformation (referred to as in the text) is shown in grey. The subdomain screw axes used to describe the transition in terms of individual subdomain rigid-body motions (see Table 3 and Text S4) are indicated; the screw axis corresponding to the C subdomain, ĉ, is omitted for clarity and shown in Figure 10E.
Figure 10.
Structural rearrangement of the myosin subdomains along the rigor-like/post-rigor NMSM path; see also the corresponding sections in the text.
The energy-minimized rigor-like structure is shown in colors; the NMSM post-rigor conformation is in grey with the nucleotide-binding elements shown in pale colors. The color code for the motor subdomains is the same as in Figure 2. The red arrows indicate the direction of motion of the subdomains along the NMSM transition. Insets on the left-hand side of each panel help to localize the structural elements which are being discussed. (A) N/U50 subdomains. Large-amplitude rotation of the N and U50 subdomains coupled to a local rearrangement in the ATP binding site; the screw axes û and nˆ are shown. (B) Structural transition of the nucleotide-binding elements. The two perpendicular views show the way switch I approaches the P-loop and moves “over” it. In doing so, the distance between Ser 218 and Thr 170, and Glu 204 and Lys 174 is substantially reduced (top), as reported in Table 4. In the ATP-bound state, the former pair of residues contributes to the coordination of the Mg2+ ion, while the latter pair makes a salt-bridging interaction. (C) N/C subdomains. On top is shown the network of H-bonds at the N/C interface responsible for the coupling. On bottom is shown the large-amplitude rotation of the N subdomain promoting the repositioning of the converter. The movement of the N-terminal is transmitted to the converter by specific interactions (shown as cyan dashed solid lines) involving Loop 76–81 (in violet). (D) L50/C subdomains. On the left are shown the large-amplitude motions of the converter and the L50 subdomain, which contribute to the opening of the U50/L50 cleft; on the right are shown the specific interactions involving the relay helix responsible for the L50/C coupling (on top) and the effect of the motion of L50 on the position of switch II (on bottom). The large-amplitude rotation of the L50 subdomain, which completes the opening of the U50/L50 cleft, is coupled to a rigid-body movement of switch II that breaks the rigor-like H-bonding interaction (Phe 441 - Ala 684) with the SH1 helix (see Table 4). (E) Reorientation of the lever arm. The lever arm, the relay helix, and the SH1 helix are shown in yellow, red, and magenta, respectively. Along with the orthogonal view (see panel C, on bottom), the picture shows how the displacement of the N subdomain is transmitted to the converter and transformed into a torque about axis ĉ that reorients the lever arm. The analysis suggests that the “short swinging” of the lever arm as observed in the X-ray structures [36] is a consequence of the rigor-like/post-rigor displacement of the converter.
Table 4.
Observables that serve to monitor the allosteric mechanism which links ATP-binding to the opening of the U50/L50 cleft.
Figure 11.
Structural rearrangement of the 7-stranded β-sheet.
(A) Side and top views of the central β-sheet are shown on the left- and right-hand side of the panel, respectively. Individual β-strands, βi, are represented by red and black arrows indicating the direction of the rigor-like and NMSM post-rigor β-strand vectors, respectively. In the box diagrams, the twist of the entire sheet, τ, and the difference in the twist angle between the individual strands and β7, Δτi,7, relative to the rigor-like conformation are monitored along the NMSM path. The former is computed as , where
and
are the unit vectors corresponding to the structural borders of the β-sheet; the latter as Δτi,7(ξ) = ∥τi,7(ξ)−τi,7(0)∥, where ξ = 0 indicates the rigor-like conformation and ξ = 1 the NMSM post-rigor conformation. (B) The comparison of the NMSM post-rigor conformation with the structure obtained by the pure rigid-body motion described by the screw axes given in Table 3; see text. The inset on the left shows the structural location of the central β-sheet in the myosin head.
Figure 12.
Molecular mechanism of the rigor-like to post-rigor transition as described by the NMSM pathway.
The molecular surface of the myosin subdomains is shown in tones of grey; the nucleotide is in green. Key residues at the subdomain interfaces that are responsible for the coupling between the myosin subdomains are depicted in cyan, magenta, orange, and red; they correspond to the P-loop, switch I, loop 76–81, and the relay helix, respectively. These allosteric connectors couple the local changes due to ATP binding to the more global motions of the myosin molecule. The ATP-binding signal is transmitted to the U50/L50 cleft through two distinct communication pathways (shown as heavy green lines; the large dot indicates the approximate origin of the signal). Path a involves the U50 subdomain and is consistent with the interpretation of three-dimensional electron cryo-microscopy reconstructions by Holmes et al. [60]. Path b involves the N-terminal subdomain, the converter and the L50 subdomain. In the latter, the transmission of the ATP binding signal is the consequence of the coordinated movement of the three subdomains described by the NMSM path (see Video S1). The allosteric communication results in the opening of the U50/L50 cleft and the uncoupling of the converter from the motor head.