Molecular Simulations Suggest a Force-Dependent Mechanism of Vinculin Activation  Li Sun, Jeffrey K. Noel, Herbert Levine, José N. Onuchic  Biophysical Journal  Volume 113, Issue 8, Pages 1697-1710 (October 2017) DOI: 10.1016/j.bpj.2017.08.037 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Crystal structures of (A) native vinculin and (B) D1 in complex with a VBS3 peptide. (A) Shown here is native vinculin in its autoinhibited conformation (PDB: 1TR2). Individual domains D1–D4 and Vt are labeled and shown in different colors. (Blue beads) Seven residues of Vt that constitute the binding interface exposed to F-actin are as suggested in (27). (Red traces) Shown here are the first two helices α1 and α2 of D1 that open up for full VBS3 insertion. (B) Given here is vinculin domain D1 in complex with VBS3 (PDB: 1RKC). The N-terminal bundle of D1 is in a helical-bundle converted structure to accommodate full VBS3 insertion. Protein structures were rendered with the software VMD. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 The model system in cartoon representation with reaction coordinates illustrated. The molecular system consists of a full-length vinculin molecule and a VBS3 peptide. Individual vinculin domains and VBS3 are labeled and colored consistently with Fig. 1. Two-bundle domains (D1–D3) and one-bundle domains (D4 and Vt) use different representations. Reaction coordinates are illustrated and labeled. Main reaction coordinates are labeled in magenta: ND1-VBS3, cutoff-based number of contacts formed on the D1-VBS3 interface; RMSD1–130, root-mean-square deviation of the backbone atoms of the N-terminal bundle of D1 from its native conformation; QD1-Vt, continuous fraction of contacts formed on the D1–Vt interface; and QD4-Vt, continuous fraction of contacts formed on the D4-Vt interface. QD1-Vt and QD4-Vt are defined for their respective binding interface to be effective for both contact formation in bound states and distance in dissociated states. Auxiliary and umbrella coordinates are also labeled: Xpull, the vector connecting VBS3 and the exposed actin binding interface of Vt projected onto the pulling direction; and DistA, DistB, and DistC, umbrella coordinates accounting for separation of the D1-Vt, D4-Vt, and D1-VBS3 interfaces, respectively. They are defined as distances between three pairs of Cα atoms from vinculin residues 25 (D1) and 945 (Vt) (DistA), vinculin residues 775 (D4) and 973 (Vt) (DistB), and vinculin residue 50 (D1) and the 13th residue of VBS3 (DistC). Coordinates are illustrated for (A) autoinhibited native vinculin and a dissociated VBS3, and (B) active vinculin with VBS3 fully bound. Some coordinates are shown in both states but have different typical values: (A) RMSD1–130 ≈ 0.15 nm, QD1-Vt ≈ 0.5, and QD4-Vt ≈ 0.5; and (B) RMSD1–130 ≈ 0.3 nm, QD1-Vt ≈ 0, and QD4-Vt ≈ 0. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Free-energy profiles for vinculin activation by VBS3 alone, under zero force. Each 2D profile uses a coordinate monitoring D1-VBS3 association (ND1-VBS3 or RMSD1–130) and a coordinate monitoring Vh-Vt dissociation (QD1-Vt or QD4-Vt). Four states N, I, C, and D are labeled. Initial bimolecular binding is marked with a vertical double asterisk. Helical bundle conversion TS0 and activation transition TS1 are marked with asterisks. ND1-VBS3 and RMSD1–130 are similar except that ND1-VBS3 is able to distinguish between states N and I (A and C) whereas RMSD1–130 is not (B and D). Color bars: free energy in units of kBT. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Vinculin activation by VBS3 under 40 pN force may proceed along one of two pathways. (A and D) Given here are 2D free-energy profiles, each using a coordinate QD1-Vt or QD4-Vt characterizing one of the two head-tail interfaces. Transitions are marked with asterisks (TS0 and TS1 for Pathway 1 and TS2 for Pathway 2) and two activation pathways are indicated by arrows (blue for Pathway 1 and red for Pathway 2) of varying width. Two arrows for the same pathway are kinetically related approximately by the positions of their bottlenecks. (B and E) Given here is a kinetic trajectory activated along Pathway 1. For clarity, only the trajectory segment near the activation transition is shown. Time sequences are indicated by a continuous variation of colors from blue to red. The transition path is highlighted in dark magenta. (C and F) Shown here is a kinetic trajectory activated along Pathway 2. Color bars: free energy in units of kBT. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Vinculin activation Pathway 1 is rate-limited by disrupting the D1-Vt interface and has low sensitivity to force. (A) Effective regions for helical bundle conversion TS0 (black ‡) and activation transition TS1 (blue ‡) in dashed rectangles are illustrated on a 2D free-energy profile under 0 pN force (Fig. 3 A). Color bar: free energy in units of kBT. (B) Free-energy profiles on ND1-VBS3 indicate low barriers for TS0 and metastability of state C. (C) Free-energy profiles on QD1-Vt indicate that the rate-limiting transition TS1 has limited sensitivity to force. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Vinculin activation Pathway 2 is accelerated by force. Free-energy profiles use coordinate qrmsd,4 = (RMSD1–130,QD4-Vt) (A and B) or qrmsd,pull = (RMSD1–130,Xpull) (C and D), at force 20 pN (A and C) or 40 pN (B and D). The coordinate qrmsd,pull contains a pulling component Xpull and displays the activation transition TS2 at 20 pN (C) that is poorly defined by qrmsd,4 (A). The activation barrier is lowered by force, and basin I moves along Xpull with force toward TS2 that stays fixed (C and D). TS2 is marked with an asterisk for qrmsd,4 (A and B) and a ‡ for qrmsd,pull (C and D) to indicate the kinetic relevance of qrmsd,pull. Color bars: free energy in units of kBT. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Histogram of vinculin activation along Pathway 1 (blue) or Pathway 2 (red) with 50 independent trajectories at each force. Longer trajectories are used for low forces to obtain sufficient statistics. Two force regimes are defined. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 8 Cartoon illustration of vinculin activation pathways regulated by force. Stable states include native state N, inactive intermediate state I, helical-bundle converted state C, and dissociated active state D. Transition states include TS1 and TS2. The notation “∼” is used to indicate the presence of F-actin association (I˜, C˜, D˜, TS˜1, and TS˜2) but does not distinguish the state from its counterpart without F-actin. Step 1: VBS3 partially binds D1. Step 2: F-actin retrograde flow engages vinculin by transient associations with an actin binding site exposed on Vt. Step 3: VBS3 fully inserts into D1 and causes a slight destabilization of the D1-Vt interface; this step contains a helical bundle conversion TS0 in the N-terminal bundle of D1. Steps 4 and 5: Vinculin activation TS1 is triggered by substantial destabilization of the D1-Vt interface. Steps 6–8 are similar to steps 3–5. Vinculin activation Pathway 1 follows steps 3–5 under zero force or steps 6–8 under low force. Steps 9 and 10: Along Pathway 2 under high force, vinculin activation TS2 is triggered by substantial release of Vt from the pair of pincers formed by D1–D3. A second actin binding site on Vt originally masked by Vh–Vt interactions is exposed and the molecular link is strengthened. Steps 11 and 12: When tension is released, vinculin refolds to its autoinhibited conformation by a mechanism that remains unclear. To see this figure in color, go online. Biophysical Journal 2017 113, 1697-1710DOI: (10.1016/j.bpj.2017.08.037) Copyright © 2017 Biophysical Society Terms and Conditions