SMD simulations of cadherin-23 with modified linkers and Ca2+-binding assays. (A-C) Force applied to N-terminus versus end-to-end distance for stretching simulations of wild-type cadherin-23 EC1+2 with Ca2+ at sites 0, 2, & 3 and Na+ at site 1 (A, S6c-g); Ca2+ at sites 0 & 3 and Na+ at sites 1 & 2 (B, S11b-f), and all binding sites empty (C, S9b-f). Colors denote independent simulations using different stretching speeds and thermodynamic ensembles as in Figure 3C. See also Figure S2. (D) Force applied to N-terminus versus end-to-end distance for simulations of EC1+2 D101G with Ca2+ at sites 0, 2, & 3 and Na+ at site 1. (E) Force peak maxima for cadherin-23 EC1+2 simulations versus stretching speed. Force peaks of simulations with Ca2+ at least at sites 0, 2, & 3 are red (S12b-f), maroon (S6c-g), yellow (S17c-g; D101G), and orange (S15b-f; CUT); with Ca2+ at sites 0 & 3 and Na+ at sites 1 & 2 are green (S11b-f) and dark green (S22b-f; D101G); with all Ca2+-binding sites empty are blue (S9b-f) and violet (S20b-f; D101G). (F) Force peak maxima for cadherin-23 EC1+2 when force is applied to center of mass of Cα1-5;36-41;86-89 atoms at the N-terminus and Cα118-121;171-173;176;203-205 at the C-terminus. Peak values color-coded as in (E), corresponding to simulations with Ca2+ at least at sites 0, 2, & 3 (red, S12g-k; maroon, S6h-l; yellow, S17h-l; orange, S15g-j); with Ca2+ at sites 0 & 3 and Na+ at sites 1 & 2 (green, S11g-j; dark green, S22g-j); and with all Ca2+-binding sites empty (blue, S9g-k). Logarithmic regression fits are shown for each simulation set. (G) Fluorescence vs added Ca2+ in competition assays between the Ca2+ chelator mag-fluo-4 and the wild-type cadherin-23 EC1+2 (black) or the D101G EC1+2 mutant (blue). Data were fitted using a four-binding-site model (Table S7). (H) Trypsin digests of wild-type and D101G mutant cadherin-23 EC1+2 analyzed by SDS-PAGE. Incubation in a range of Ca2+ concentrations shows different Ca2+-dependence of proteolysis protection. The intact proteins migrate at 25 kDa. (I) Quantification of the intact protein in the gels presented in H. Fits using the Hill equation indicate an effective KD of 86.8 μM for wild-type and 470.4 μM for the D101G mutant (Hill coefficients: 3.7 and 1.3, respectively). See also Figures S4, S5, and S6.