Direct observation of a coil-to-helix contraction triggered by vinculin binding to talin

Figures

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  Fig. 1. Vinculin binding requires the structural accommodation of the talin polypeptide on the vinculin head: Under force, talin unfolds and the vinculin-binding sites become unstructured polypeptide chains. Upon binding of one vinculin head molecule, the binding site helix reforms, which shortens talin by ∼1.5 nm at a force of 9 pN.

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  Fig. 2. Real-time detection of the coil-to-helix contraction induced by vinculin head binding. (A) Schematics of a magnetic tweezers experiment for detecting vinculin binding events. We engineer an (R3)-(I91)8 protein construct, flanked by a HaloTag for covalent tethering to a glass coverslip, and a biotin for anchoring to streptavidin-coated superparamagnetic beads. Physiological-level forces in the piconewton range are applied through a magnetic field gradient created by either a pair of permanent magnets or a magnetic head. The experiment is conducted in the presence of vinculin head, and the extension changes due to folding or binding are measured with nanometer resolution. (B) Magnetic tweezers recording showing individual vinculin head binding events to the R3 IVVI domain. At 9 pN, R3 IVVI folds and unfolds in equilibrium, which yields extension changes of ∼20 nm. In the presence of 20 nM vinculin head, these dynamics eventually stop due to the binding of vinculin head. This event is resolved as a ∼3-nm contraction that occurs in the unfolded talin polypeptide due to the reformation of the α-helices of its two vinculin-binding sites (red arrow, inset). The complex dissociates at…

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  Fig. 3. Vinculin head binding contracts unfolded R3 IVVI. (A) Averaged recordings of the binding contractions at different pulling forces. The magnitude and duration of the contraction depend on the force. Traces averaged from >10 recordings. (B) Unbinding steps at different pulling forces. Two ∼3-nm steps are observed, after which talin recovers its ability to refold. (C) Average step sizes for the binding contractions (red) and unbinding steps (blue) measured as a function of the pulling force. The binding contraction scales with force following the FJC polymer model with a contour length of 7.3 nm, which agrees with the simultaneous formation of the two α-helices of the vinculin-binding sites. The steps of unbinding have half that contour length, indicating that they correspond to the unraveling of a single binding site helix. Error bars are the SEM; data collected over 35 molecules, 156 binding steps, and 501 unbinding steps.

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  Fig. 4. Stoichiometry of vinculin head binding to R3 IVVI. (A) Magnetic tweezers recordings of R3 IVVI in the presence of 8, 30, and 80 nM vinculin head at a force of 9 pN. The waiting time for vinculin binding (tb) can be measured at the single-molecule level as the time taken since the probe force is set until the contraction is observed and the hopping dynamics stop. (B) Distribution of vinculin head binding times at different concentrations, and a force of 9 pN. The shape of the distribution follows a single-molecule enzymatic model, where two vinculin head molecules bind simultaneously to unfolded R3. (C) Concentration dependence of the rate of vinculin head binding, which follows a second-order Hill-like equation. This demonstrates that two vinculin head molecules bind simultaneously to the unfolded talin R3 domain. The point at 0 nM was estimated from three very long recordings in the absence of vinculin head (5, 7, and 36 hours) that showed no arrest of talin folding dynamics. Vertical error bars are the SEM, and horizontal error bars are the precision on the determination of the vinculin head concentration (12.3%). Number of events measured: 8 nM, N = 122; 20 nM, N = 184; 30 nM,…

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  Fig. 5. Mechanical force regulates vinculin head binding. (A) Typical recordings of vinculin head binding to R3 IVVI at different forces during a 50-s time window at a concentration of 20 nM. (B) Binding probability measured over a 50-s time window as a function of force. Force has a biphasic effect on binding, favoring it by unfolding talin, but hampering it due to the energy penalty of the coil-to-helix contraction. The data are described by a simple model based on this mechanism (dashed lines; see section X in the Supplementary Materials). Errors are SEM; data collected over 20 molecules and 259 observations for R3 IVVI, and 10 molecules and 213 observations for R3 WT.