Massively parallel mapping of substrate cleavage sites defines dipeptidyl peptidase four subsite cooperativity

Substrate specificity determines protease functions in physiology and in clinical and biotechnological application. However, affordable and unbiased assays for large-scale quantification of substrate cleavage have been lacking. Here, we develop hiMAPS (high-throughput mapping of protease cleavage sites), a cheap, mass spectrometry-based assay, to derive cleavage motifs for human Dipeptidyl Peptidase Four (DPP4), a key regulator of blood glucose levels. We recapitulate the known benefit of proline in the penultimate (P1) position from the substrate N-terminus, extend it to additional residues, and identify and quantify combinatorial interactions of P1 with its neighbors. These findings reveal extensive cooperativity among the enzyme's active site subsites and allow us to derive a sequence motif that predicts substrate turnover. We show that this information provides new opportunities to engineer stabilized versions of a key substrate of DPP4, the small peptide hormone GLP-1, whose derivatives are used for clinical treatment of type 2 diabetes and obesity. Finally, structural and biochemical characterization of a DPP4 homologue, C. elegans DPF-3, reveal specific subsite differences and a distinct cleavage motif, providing insight into the mechanistic basis of the observed specificity. Collectively, our findings present a broadly applicable framework to high-throughput mapping of protease cleavage sites, extend our understanding of protease specificity, and provide a chemical space for substrate engineering of a clinically relevant family of exopeptidases. ### Competing Interest Statement The authors have declared no competing interest.