A direct computational assessment of vinculin–actin unbinding kinetics reveals catch-bonding behavior

Figures

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  Fig. 1.. (A) Illustration of the two systems under study. It is hypothesized that upon binding actin (light green) the vinculin tail domain (Vt) converts from a five helical state to a four helical state, with the H1 helix being unresolved in the experimental structure. (B) Overlay of two Actin-Vt models. The “Aligned” (aVt, colored red) configuration contains helices H1–H5 while “Holo” (hVt, blue) has only H2–H5. Actin subunits A1 and A4 (colored gray) are restrained in the MD simulation to maintain the helical structure and prevent rotation. Arrows show primary collective variables (CVs) studied, Q‖ and Q⊥, which characterize movement of vinculin along and perpendicular to the filament, respectively. These CVs are defined using the vectors v→12, which point between the center of mass (COM) of actins A1 and A2 (p1) and the COM of actins A4 and A5 (p2), and v→13, which points from p1 to the COM of Vt helices H2–H5 (p3). (C) Superposition of the Aligned and Holo configurations in an equilibrated configuration shown separately and as in their binding pocket, with α-helices and C-terminal extension (CTE) labeled. This superposition is also shown in the Movie S1. (D) Plot shows approximate…

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  Fig. 2.. (A) FES for the Holo system (hVt) computed by 2D OPES-MetaD biasing Q‖ and Q⊥. The string shows the minimum free energy path on this surface going from the bound to unbound state. (B) Same as in A, for the Aligned (aVt) system. (C) One-dimensional projections of the minimum free energy paths from (A and B). The Q⊥ and Q‖ values at the putative transition state are labeled; gray lines indicate points before the transition state and are used subsequently to help define the excluded region in unbinding lifetime calculations. Error bars were computed by bootstrapping the aggregated CV trajectories (Section 4.2).

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  Fig. 3.. (A) 2D FES for Holo and Aligned as shown in Fig. 2, now with applied forces along Q‖ of ±30 pN, illustrating how applied force tilts the FES to larger or smaller values of Q‖. (B) Final snapshots of unbound states produced by positive and negative pulling forces of magnitude 30 pN along Q‖. (C) Transition barriers ΔA‡ along minimum free energy paths for Aligned and Holo. Holo shows an increase in barrier in both directions with force, with a slightly larger increase for negative F, while Aligned shows an increase only for negative F.

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  Fig. 4.. Lifetimes of Vt unbinding extracted by OPES-Flooding for Holo (Top) and Aligned (Bottom). Both models show asymmetric catch sensitivity to applied forces along Q‖ in this physiological force regime. These lifetimes were obtained by fitting cumulative distribution functions as shown in SI Appendix, Fig. S12. Error bars were computed by bootstrapping as described in Section 4.3.

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  Fig. 5.. (A) Snapshots showing the initial configuration of our Holo+H1 system, and final configurations after pulling on the N-terminus at ±50 pN along Q‖. Pulling the N-term toward the pointed end (+50 pN) docks H1 along the bundle, while pulling toward the barbed end (−50 pN) extends H1 away from the rest of Vt. (B) Histogram of the distance between the COM of H1 and the COM of H2–H5 under force. The Aligned system (gray) maintains a distance close to the crystal structure (dashed line), while pulling with positive force (blue) and negative force (orange) result in more or less association, respectively. (C) Histogram of the number of contacts (atom pairs with distance below 5 Å) between H1 and H2–H5 using atoms carbon oxygen and nitrogen. (D) Histogram of the Cα RMSD between helices H2–H5 in Holo+H1 and H2–H5 in the PDB structure 1QKR.

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  Fig. 6.. Representative structures of Vt bound to actin, with hVt in blue, aVt in red, and actin in green. The initial structure (6UPW) is overlaid on the Holo structures after superimposing actin A3 from the MD and EM structures. Residues in Vt and actin A3 and A5 separated by less than 3 Å are shown in VMD’s (63) “licorice” representation. (A and B) Representative structures from 500 ns of equilibrium MD on Holo and Aligned systems. (C and D) Representative structures from OPES-MetaD simulations of Holo with and without force illustrate the primary changes induced by pulling on Vt’s COM.