Biochemical and Structural Studies of the Midnolin Catch Domain Bound with Both Wild-Type and Mutant IRF4 Peptides Reveal the Molecular Basis for Its Broad Substrate Specificity

• The crystal structure of the midnolin Catch domain bound to IRF4 peptide reveals a substrate-binding groove formed at the interface of Catch1 and Catch2 subdomains, which accommodates a β-strand from the substrate, forming an antiparallel five-stranded β-sheet. • The Catch domain recognizes a specific sequence motif (x[G/A/V/L/I/M/P/F/Y/C/S/T]x[G/S]x[F/Y]x[G/A/V/L/I/M/P/F/Y/C/S/T]x) in substrates, with hydrophobic interactions and spatial complementarity being the primary determinants of binding specificity. • Mutational analysis shows that positions V2 and A8 in the IRF4 motif tolerate only hydrophobic or small polar residues, while G4 can be replaced by Ser and F6 by Tyr, indicating a broad but selective substrate repertoire. • The findings provide a molecular basis for midnolin's broad substrate specificity, enabling recognition of diverse substrates with unstructured or loop regions containing the G/SxF/Y motif. • This study advances understanding of the ubiquitin-independent proteasomal degradation pathway mediated by midnolin, which is crucial for cellular protein homeostasis and has implications for diseases like Parkinson's and cancer.