• MIDN recognizes substrates via a conserved β-strand insertion mechanism at the Catch1-Catch2 interface, enabling broad specificity.
• A minimal 'F-G zipper' interaction between substrate and Catch2 is the dominant energetic determinant for binding.
• Flanking residues tolerate substitution due to large plastic hydrophobic pockets, explaining substrate diversity.
• A consensus motif (G/S-x-F/Y) provides a predictive framework for identifying new MIDN targets, with implications for immune regulation, neurodegeneration, and cancer.