(A) Domain organization of CAPN7 and IST1, depicting the binding interaction between tandem MIT domains of CAPN7 and MIT-interacting motif (MIM) elements of IST1 (red double-headed arrows). Domain definitions: CORE, helical ESCRT-III core domain of IST1 that functions in filament formation; CBSW, tandem calpain-type beta sandwich domains of CAPN7. (B) Fluorescence polarization anisotropy binding isotherms showing CAPN7(MIT)2 binding to IST1 constructs spanning tandem or individual MIM elements (IST316-366, IST1316-343, and IST1344-366, respectively). Isotherm data points and dissociation constants (KD) are averages ± standard error of the mean from three independent experiments. Error bars on the IST1344-366 isotherm are entirely masked by the data symbols. (C) NMR mapping of the CAPN7(MIT)2 binding sites on IST1303-366. Sections of overlaid HSQC spectra of free IST1303-366 (black contours) and IST1303-366 saturated with 1.3 molar equivalents of CAPN7(MIT)2 (orange contours) are shown. Amide NH resonances in the unbound state (black contours) that lack bound state resonances (orange contours) correspond to residues that experience large intensity perturbations upon CAPN7 binding (amino acid residue labels in green). In contrast, strong resonances that overlap well in both the unbound and bound states (black and orange contours) correspond to residues that experience smaller intensity perturbations upon CAPN7 binding (amino acid residue labels in black) (see Figure 1—figure supplement 2 for the entire spectra). (D) Amide intensity ratios (unbound/bound) for each residue of the IST1303-366 peptide. Small ratios (<5, 30 residues, lower dotted line) correspond to residues that remain dynamic in the complex, whereas large ratios (>15, 20 residues, upper dotted line) correspond to residues whose dynamics are reduced upon complex formation (and therefore likely contact CAPN7(MIT)2 and/or become ordered upon binding). Proline residues were not scored (asterisks). IST1 MIM elements show either the bounds of interpretable electron density from the crystal structure of the complex (top boxes, light green, see Figure 2 and Figure 2—figure supplement 1) or the bounds of the complex as defined by NMR resonance intensity changes (bottom boxes, dark green, see (C) and Figure 1—figure supplement 2).