De novo design of metalloproteases for targeted amyloid-β cleavage

De novo protein design has not yet achieved the creation of proteases capable of selectively cleaving any desired peptide bond within a native protein with high precision. Here, we report the use of the flow-based generative model Proteus2 to design metalloproteases by generating enzyme-substrate complexes conditioned on a target peptide sequence and a predefined catalytic motif. Our approach employs a two-step encapsulation strategy to create clamp-like metalloproteases that bind the target peptide in a manner that maximizes substrate sequence specificity. The generative process simultaneously optimizes the precise positioning of the target peptide bond in a catalytically competent configuration and accurately scaffolds the transition state catalytic residues-both essential for specific and efficient catalysis. Using this strategy, we designed zinc metalloproteases targeting three distinct cleavage sites within the aggregation-prone regions of amyloid-{beta} (A{beta}), a key pathogenic factor in Alzheimers disease. Experimental characterization validated five enzymes, each capable of precise cleavage at the intended sites with high specificity and minimal or undetectable activity on non-cognate substrates. On average, these enzymes accelerated peptide bond hydrolysis by more than 107-fold relative to the uncatalyzed reaction, and enabled efficient digestion of the A{beta} peptide into smaller segments when enzymes targeting different sites were combined. Cryo-EM structures of three designed enzymes in complex with A{beta} peptide, each targeting a distinct cleavage site, revealed close agreement with the design models. Together, these results demonstrate the potential of sequence-guided, generative approaches for developing programmable, sequence-specific proteolysis and lay the groundwork for future applications in basic research and therapeutic development.