1. Volume 25, Issue 12, Pages 1856-1866.e2 (December 2017)
Pressure-Dependent Chemical Shifts in the R3 Domain of Talin Show that It Is Thermodynamically Poised for Binding to Either Vinculin or RIAM 
Nicola J. Baxter, Thomas Zacharchenko, Igor L. Barsukov, Mike P. Williamson 
Structure 
Volume 25, Issue 12, Pages e2 (December 2017)
DOI: /j.str
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2. Structure 2017 25, 1856-1866.e2DOI: (10.1016/j.str.2017.10.008)
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3. Figure 1
Model for the Role of Talin in the Formation of Focal Adhesion Complexes
Talin is a long rod-like protein. One end contains an integrin binding site (IBS) while the other contains an actin binding domain (ABD). At rest, RIAM binds to the closed R3 domain of talin and anchors it to the cell membrane. When actin filaments are pulled, the R3 domain undergoes a conformational change, which causes RIAM to dissociate and vinculin to bind. Vinculin attaches talin to the actin cytoskeleton and thereby stabilizes the focal adhesion complex. Figure adapted from Klapholz et al. (2015).
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4. Figure 2
15N-HSQC Spectra of R3 and R3-IVVI Acquired at Pressures from 1 bar (Red) to 2.5 kbar (Violet)
R3 (A) and R3-IVVI (B). The insets show the pressure-induced changes in backbone amide peak position for G796 and D799, positioned at the N-terminal end of helix 1, and V837 which is located at the center of helix 2. These residues together with others show dramatic curvature for R3, whereas their behavior is less curved for R3-IVVI. For a direct comparison of spectra of R3 and R3-IVVI, see Figure S1. For chemical shift differences and their locations on the structure, see Figure S2.
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5. Figure 3 Analysis of SVD Fitting
(A) Plot of log(σi) versus i for the SVD combined analysis of backbone amide HN and N observed chemical shift versus pressure data for R3. The value of σ11 is 0.
(B) Plot of the first 5 of the 11 column vectors of V. Vectors 1 to 4 are indicated by circles and lines colored black, blue, magenta, and red, respectively, and vector 5 is shown as cyan circles. For fitting to more simple quadratic and cubic equations and locations of poorly fitting residues, see Figures S3 and S4. For the equivalent analysis of SVD fitting for R3-IVVI, see Figure S7.
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6. Figure 4
Examples of Noise-Free Chemical Shift versus Pressure Data for R3
All curves are rescaled to a maximum chemical shift change of 100% to illustrate the variable response of specific nuclei to increasing pressure. Experimental data are indicated by circles, and the fits to Equation 2 (fitted with a global ΔG and ΔV and resonance-specific δ1o, δ2o, Δδ1, and Δδ2 parameters) are shown by lines: F813 HN (black), V823 N (blue), I828 N (green), R797 C′ (red), T809 C′ (magenta) and A877 C′ (cyan).
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7. Figure 5 TALOS-N Predictions for Backbone Dihedral Angles
ϕ (A) and ψ (B) dihedral angles of the ground state (closed conformation, blue circles) and the excited state (open conformation, red circles), compared with the ten lowest energy members of the PDB: 2L7A NMR structural ensemble (black circles). The four α helices are indicated.
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8. Figure 6
Differences in Chemical Shift (Δδ = δ1o – δ2o) between the Ground State Structure (Closed Conformation) and the Excited State Structure (Open Conformation) of R3
Cα nuclei (A) and Cβ nuclei (B). The bars indicate relative chemical shift changes from δobs for δ1o (ground state: black) and δ2o (excited state: red). The four α helices are indicated. For corresponding differences in HN, N and C′, see Figure S5, and for the differences for R3-IVVI, see Figure S8.
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9. Figure 7
Differences in Chemical Shift (Δδ = δ1o – δ2o) between the Ground State (Closed Conformation) and the Excited State (Open Conformation) of R3
Differences are shown on the lowest energy NMR structure. The disordered N terminus is not shown and the cartoon depictions comprise residues A795 to K911. The N and C termini are indicated and helices are numbered.
(A) Δδ values for Cα nuclei (colored backbone and spheres) and Cβ nuclei (spheres on side-chain sticks) with positive values in blue and negative values in red. Only differences >1 SD are indicated, with large differences (>3 SDs) in deeper colors and larger spheres.
(B) Same as (A) except that R3 is rotated 180° about a vertical axis.
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10. Figure 8
Differences in Pressure-Dependent Gradients (Δgradient = Δδ1 – Δδ2) for HN Nuclei between the Ground State (Closed Conformation) and the Excited State (Open Conformation)
R3 (A) and R3-IVVI (B) shown on the lowest energy NMR structure. The disordered N terminus is not shown, and the cartoon depictions comprise residues A795 to K911. The N and C termini are indicated, and helices are numbered. Δgradient values >1 SD are shown with positive values in blue and negative values in red, with large differences (>3 SDs) indicated as deeper colors. For a graphical view of the gradient values for R3 and R3-IVVI, see Figures S6 and S9, respectively.
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11. Figure 9 Proposed Mode of Action of Talin Domain R3
(A) Binding site on R3 for RIAM in the closed state.
(B) Binding site on R3 for vinculin in the open state.
(C) In the unactivated full-length protein, R3 is closed, and helices 2 and 3 form a binding site for RIAM, which is able to bind reversibly to the closed domain. Force, provided by movement of the actin cytoskeleton relative to talin, pulls helix 1 out from the bundle, exposing the binding site on helix 2 for vinculin. This enables vinculin to bind, further opening out the bundle, which then exposes helix 3, forming a second vinculin binding site and leading to complete opening of all four helices. The IVVI mutation stabilizes the closed state and disfavors vinculin binding.
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