Structural and Mechanistic Basis of Selective Proteolysis by Zr-Based Metal–Organic Frameworks

Zirconium-based metal–organic frameworks (MOFs) have emerged as promising alternatives to proteolytic enzymes, offering tunable platforms for selective protein cleavage with potential applications in proteomics, protein engineering, and therapeutic development. However, the limited mechanistic understanding of MOF–protein interactions and the molecular origins of cleavage specificity hinder their broader application as nanozymes. In this study, we investigate the selective hydrolysis of cytochrome c by the Zr-based MOF UiO-66, employing an integrated approach combining high-resolution mass spectrometry and molecular modeling. UiO-66 exhibits preferential cleavage at peptide bonds adjacent to glycine, threonine, and lysine residues, particularly when two or more of these residues are in close proximity, suggesting a cooperative recognition mechanism. Computational analyses reveal that glycine’s minimal steric bulk facilitates optimal alignment of the scissile bond with the Zr 6 O 8 active site, while threonine and lysine contribute to substrate binding and orientation through hydrogen bonding and electrostatic interactions with surface-exposed carboxylates on the MOF linker. This residue-specific selectivity contrasts with the aspartic acid preference observed in discrete Zr-based nanoclusters, underscoring the critical influence of the MOF’s extended 3D framework on proteolytic behavior. These findings provide mechanistic insights into MOF-mediated proteolysis and highlight the potential of framework-engineered nanozymes for targeted protein cleavage in advanced proteomic applications.