How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism
Figure 3
Fragmentation of the Talin Rod into α-Helix Subbundles Leads to the Sequential Exposure of the Vinculin Binding Helices (VB Helices)
(A) Sequential structural snapshots of the mechanically strained talin H1–H12 rod. Three intermediate states are observed (I1, I2, I3). The pulling force of 300 pN was used in the depicted SMD simulation. The extension, i.e., the increase in the length of the H1–H12 as compared to the equilibrium state which measured 3.2 nm, is shown as (ΔL). Key transitional unfolding events detected in this simulation as H9–H12 separates from rest of the protein are shown in T1 and T2.
(B) SMD simulations showed a transient bending of helix H9 at the onset of breaking the H1–H12 bundle into two pieces. The bending occurred at residue Thr772, which initially formed intrahelix bonds with Gly768:O with both the backbone nitrogen and the side chain oxygen. The hydrophilic side chain in Thr772 attracts water molecules that enter the helix bundle cleft when the talin rod is strained. The waters soon reach the helix backbone (5.514 ns), and then compete and attack the backbone hydrogen bond initially formed between Thr772 and Gly768, finally leading to the bending of the helix (5.631 ns).
(C) Constant force extension-time plots when force is distributed over the length of the terminal helices H1 and H12 (200, 250, 300, or 400 pN). The distance between Cα-atoms of residues 504 and 865 is given. The extensions of the structural snapshots shown in (B) are indicated in the extension time-plots in (C).
(D) Change in the buried surface area of the VB helices during equilibration and when extended under 300 pN force calculated for the blue 300 pN trace shown in (C). The buried areas are shown normalized to the average buried area obtained during equilibration. The lowest graph shows the average buried area of the VB helices (red) and of other helices (nonVB, black). The dotted pink lines give the times at which the non-equilibrium structural snapshots in (B) were taken. The respective points of “activation”, i.e., when the buried areas of helices H6, H9, H11, and H12 in talin equal the experimentally found buried areas of isolated talin helices in complex with the vinculin head, are given as blue asterisks. For H6 and H9, the buried area determined for the H11-vinculin complex is used as a reference because there is no available structure of those helices in complex with vinculin. The buried area of H4 was higher than the buried area of the VH-H4 complex for the whole simulation period. The buried area of a helix was calculated by measuring the solvent accessible surface area (SASA) of the helix alone and subtracting the SASA of the helix embedded in the protein by using the program VMD. The scanning probe used was 1.4 Å.
(E) Top views of the VB helices along the helix axes. Water molecules (red dots) located within 5 Å of the VB helix core (10–13 residues in the middle of the helix) from 20 frames over 100 ps time window are plotted together with the protein structure at different time points. The side chains of 10 most conserved residues in VBS helices [10], according to the consensus sequence LxxAAxxVAxALxxLLxxA are shown in a yellow stick representation.
(F) Side views showing how water penetrates into the interface between the subbundles H1–H8 and H9–H12. The water molecules located in the vicinity of residues Leu716, Leu736, and Gly740 are plotted over a 100 ps time window (20 frames). The frames in the trajectory are aligned according to H8.