Strain regulates the ADP release kinetics of the trailing head.

<p>(A) A schematic representation of myosin dimer showing how P_i release can create strain on the trailing head. This strain arises due to the postponed-powerstroke waiting state of the leading head (left head). (B) Probability distribution of distances between small and big subunits of the MH domain at different level of strain on the trailing head. This distribution shifts towards the larger distances as strain increases, which leads to an increase in ADP release rate with increasing strain.</p

Functional cycle of single- and double-headed myosin.

<p>(A) Sequence of events of the mechanochemical cycle of the single-headed myosin. ATP bound (red) head (i) binds to the actin filament followed by the hydrolysis of ATP. The arm (black line on head) of this actin bound head (ii) in ADP + P_i state (pink) has a pre-powerstroke conformation. Phosphate (P_i) release induces the powerstroke conformational change to the lever arm (iii). Next, ADP (blue) is released from the bound head while keeping the post-powerstroke lever arm conformation (iv). Finally, the empty head (gray) detaches from the actin followed by an ATP intake. The ATP dependent unbound head experiences a repriming event to its stable pre-powerstroke lever arm conformation (v). Note that, state (v) is exactly same as state (i) with an additional stepping towards left and also the nucleotide dependent actin binding affinity information in the middle. (B) The sequence of events of the functional cycle of the double-headed myosin. In state (i), head 1 is in an ATP bound state and head 2 is bound to actin in an ADP state. ATP hydrolysis and subsequent binding to actin by head 1 provide a two-head bound myosin (ii). Head 1 releases phosphate (P_i) to transform into state (iii). In contrary to a single-head myosin, here, after P_i release, head 1 cannot perform powerstroke while head 2 is still bound. The green line shows the expected lever arm conformation of head 1. It is important to note here that the two-head-bound state iii could adopt an alternative conformation with the converters of both heads in a post-powerstroke conformation while the lever arm of the leading/trailing head bends backward/forward. Next, ADP releases from head 2 (iv) and subsequently ATP binds to the empty head. Head 2 now detaches from the actin and head 1 now performs its postponed powerstroke step. Finally, head 2 also perform its spontaneous repriming event to form state (v). Note that, state (v) is exactly same as state (i) with an additional stepping towards the left.</p

Two important conformational states of the mechanical cycle of myosin.

<p>(A) Pre- (PDB 2V26 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005035#pcbi.1005035.ref034" target="_blank">34</a>]) and post-powerstroke (PDB 2BHK [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005035#pcbi.1005035.ref036" target="_blank">36</a>]) states of the myosin motor showing the motor head (MH) and the converter domains. Note the change in the conformation of the converter domain with respect to the MH in the two conformations. The competing interactions (contacts) between the MH and the converter domains, responsible for stabilizing these two states, are shown by lines. (B) Residue-residue contact map for both the conformations. The two dotted green lines are drawn to separate the contact maps of the MH and the converter domains. In the upper triangle, the post-powerstroke contact map (red) is overlaid upon the pre-powerstroke contact map (blue) to identify the exclusive converter-MH contacts in the pre-powerstroke state (shown by lines in Fig 2A). In contrast, the same data have been overlaid in opposite order (blue upon red) in the lower triangle to extract the exclusive converter-MH contacts in the post-powerstroke state (those interactions are shown by lines in Fig 2A).</p

Structural adaptation of the trailing head signals faster ADP release.

<p>(A) Myosin MH domain structure showing the big (red) and small (green) subunits. (B) Nucleotide binding region of the MH domain is shown in terms of P-loop (blue), switch I (red) and switch II (green). (C) RMSD distribution (P(RMSD)) of the small subunit of the MH domain after least square fitting of the big subunit from the leading and trailing head simulations. The RMSD is calculated with respect to the pre-powerstroke MH conformation. Note the larger RMSD for the trailing head indicating substantial structural changes. (D) Distribution of distances between P-loop and switch I (P(d_SWI-Ploop)) for the leading and trailing head simulations. (E) Distribution of distances between P-loop and switch II (P(d_SWII-Ploop)) for the leading and trailing head simulations. (F) Distribution of distances between switch I and switch II (P(d_SWI-SWII)) for the leading and trailing head simulations. A larger distance between switch I and switch II for the trailing head simulation compared to leading head signals faster ADP release.</p

Powerstroke of the myosin motors.

<p>(A) Powerstroke step with the release of P_i. The residues involved in the P_i mediated interactions in terms of p-loop (blue), switch I (red) and switch II (green) are shown. (B) RMSD (with respect to pre-powerstroke crystal structure) distribution for the simulation with P_i mediated interaction. The population of the pre-powerstroke ensemble is higher. (C) RMSD (with respect to pre-powerstroke crystal structure) distribution for the simulation without P_i mediated interactions. Note the inversion of the distribution with phosphate release making post-powerstroke ensemble as the dominant one.</p

Structural comparison of kinesin-1 and Ncd.

<p>(a) Dimer structures of kinesin-1 (PDB ID 3KIN) in ADP-ADP state in the absence of MT. (b) Dimer structure of the symmetric Ncd dimer (PDB ID 1CZ7). Note that the Ncd motor lacks the neck-linker region. (c) Superposition of the MHs of kinesin-1 (red) and Ncd (blue). Between the MHs of the two motors Cα-rmsd is 1.06 Å, and there is 41% sequence identify. (d) Native contact maps of the two motors, upper left for Ncd and lower right for kinesin-1. Note that the patterns of the contact map between the two MH domains enclosed in black box are very similar, and that the differences are in structural motifs peripheral to MH.</p

ATP dependent population change and minus-end directed power stroke of Ncd on MT track.

<p>(a) Distributions of the angle (θ), among LEU296 in the unbound monomer and D424, E567 in the bound monomer, calculated from simulation for φ-ADP state on MT. The structure of Ncd with <i>θ</i>∼25° (peak position) is depicted on the MTs. (b) The angle (θ) distribution calculated from simulation for the ATP-ADP state. The structure of the Ncd on the MT with <i>θ</i>∼150° (peak position) is shown on the top. Change from φ-ADP to ATP-ADP state (a→b) implies the minus-end-directed power stroke.</p

Conformational transition between symmetric and asymmetric conformers of Ncd dimer in the absence of MTs.

<p>(a) Ncd motor in the symmetric and asymmetric states. (b) Head-stalk contacts present in the symmetric (green) and asymmetric state (red) shown for a monomer. These two sets of contacts cannot be satisfied simultaneously. Residue contact maps for (c) symmetric and (d) asymmetric conformations. The difference in the head-stalk contacts in the asymmetric conformer is highlighted in the contact map by using circles.</p

Dynamics between symmetric and asymmetric states of Ncd in solution.

<p>(a) Distribution of the inter-residue distance between E567 of the two monomers calculated from simulation show two peaks corresponding to the symmetric and asymmetric states with representative Ncd structures for each peak position. (b) Two dimensional free energy diagram color-coded in <i>k</i>_B<i>T</i> unit. Energetically possible transitions are between symmetric and one of the two asymmetric states (A or B). (c) Low frequency modes from PCA lend support to the distortion dynamics that drives the Ncd dimer from its symmetric to its asymmetric state. Residue-wise vector represents one of the two low frequency modes.</p

Mechanochemical cycle for Ncd motor.

<p>In solution, Ncd exists in (i) symmetric and two asymmetric states (either (ii) or (ii)’). On the MTs, Ncd in φ-ADP state exists predominantly in (iii) symmetric plus-end directed conformer. ATP binding stabilizes (iv) asymmetric conformer, which gives rise to minus-end-directed stroke. Finally, the ATP hydrolysis returns the Ncd to the ADP-ADP state that has weak MT-binding affinity; thus Ncd dissociates from MT to the solution. The free energy (F(z)) profile of the power stroke step which is calculated by taking stalk tip position (z) on MT as order parameter is also shown in the same figure.</p