• MIDN recognizes substrates via a conserved β-strand insertion mechanism at the Catch1/Catch2 interface.
• An '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, enabling broad substrate specificity.
• A consensus recognition motif (G/S-x-F/Y) is derived, providing a predictive framework for identifying MIDN targets.