Monomeric and dimeric conformation of the vinculin tail five-helix bundle in solution studied by EPR spectroscopy

Abstract

The cytoskeletal adaptor protein vinculin plays an important role in the control of cell adhesion and migration, linking the actin cytoskeleton to adhesion receptor complexes in cell adhesion sites. The conformation of the vinculin tail dimer, which is crucial for protein function, was analyzed using site-directed spin labeling in electron paramagnetic resonance spectroscopy. Interspin distances for a set of six singly and four doubly spin-labeled mutants of the tail domain of vinculin were determined and used as constraints for modeling of the vinculin tail dimer. A comparison of the results obtained by molecular dynamic simulations and a rotamer library approach reveals that the crystal structure of the vinculin tail monomer is essentially preserved in aqueous solution. The orientation of monomers within the dimer observed previously by x-ray crystallography agrees with the solution electron paramagnetic resonance data. Furthermore, the distance between positions 1033 is shown to increase by >3 nm upon interaction of the vinculin tail domain with F-actin.

Figure 1

(A) Vt structure with R1 side chains bound to positions 901, 922, 950, 957, 984, and 1033 (PDB-code: 1ST6). (B) EPR spectra of the spin-labeled Vt constructs obtained at room temperature. (C) Plot of 〈H²〉⁻¹ (inverse of the second moment) versus ΔH₀⁻¹ (inverse of central line width) extracted from the room temperature EPR spectra of the single mutants Vt901R1, Vt922R1, Vt950R1, Vt957R1, Vt984R1, and Vt1033R1.

Figure 2

Interspin distances between R1 side chains in doubly spin-labeled Vt. (Left) Populated rotamers for Vt901R1/957R1 (H1/H3), Vt922R1/957R1 (H2/H3), Vt901R1/1033R1 (H1/H5), and Vt984R1/1033R1 (H4/H5) (stick representation) are shown attached to the Vt crystal structure (ribbon style). (Center) Distance distributions, P(r), for the doubly spin-labeled Vt constructs (shown on the left) were determined by X-band EPR spectroscopy (shaded) considering dipolar interaction only (see text). The rotamer library approach (broken lines) and MD simulations (solid) are shown for comparison. (Right) Experimentally obtained low temperature cw-EPR spectra (solid) of Vt922R1/957R1 (H2/H3), Vt901R1/1033R1 (H1/H5), and Vt984R1/1033R1 (H4/H5), as well as the DEER spectrum for Vt901R1/957R1 are overlaid with fits (shaded) calculated using programs SHORT DISTANCES and DEERAnalysis, respectively.

Figure 3

DEER spectroscopy of singly spin-labeled Vt constructs. (Left) Dipolar evolution functions (shaded) and the corresponding fits calculated with DEERAnalysis (solid). (Right) Experimental distance distributions, P(r), obtained by DEERAnalysis (solid) are compared to calculated interspin distances of R1 side chains in the dimer crystal structure (1QKR) using the RLA (shaded). The interspin distance between positions 1033 was also determined in Vt1033R1 bundling actin filaments (VTA1033R1) (dashed).

Figure 4

Crystal structure of the Vt dimer (1QKR) showing interspin distance vectors that connect Cβ positions of the spin-labeled sites used in this study.