Mechanism of talin-induced topology change. (A) Crystal structure of talin2 F2-F3 (yellow) bound to integrin β1D tail (red; Protein Data Bank accession no. 3G9W). The amino acids in talin2 that bind to the membrane-proximal β1 tail (L328) and to lipid (K325, K258, K274, R276, and K280) are indicated with corresponding talin1 residues in parentheses. β1D(I757) (corresponds to β3(I721)), the position of bimane labeling, is indicated. (B) The emission spectra of L694C-bimane nanodiscs in the presence of increasing concentrations of THD (left) or THD(L325R) (right). wt, wild type. (C) Effect of THD(K322D), which blocks lipid binding, on the emission spectra was analyzed as in B. (D) Effect of mutations in lipid-binding residues of the THD F2 domain (K256E, K272E, K274E, and R277E) on the emission spectra was analyzed in as in B. (E) I721C-bimane nanodiscs were assembled with a 1:1 mixture of DMPC and DMPG (left) or with DMPC only (right), and their responses to the addition of THD were analyzed as in B. (B–E) Data are representative of at least three independent experiments.