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 midnolin Catch domain recognizes a specific sequence motif (G/SxF/Y) in substrates, enabling broad specificity through hydrophobic interactions and spatial complementarity. • Crystal structure of Catch-IRF4 complex reveals a substrate-binding groove formed by Catch1 and Catch2 subdomains, with the IRF4 motif forming a β-strand that extends an antiparallel β-sheet. • Mutational analysis defines permissible substitutions at key positions (V2, A8, G4, F6) of the IRF4 motif, excluding large polar and charged residues, which guides substrate prediction. • These findings provide a molecular basis for the ubiquitin-independent proteasomal degradation pathway mediated by midnolin, with implications for understanding protein homeostasis and potential therapeutic targeting.