Structural and proteomic analysis of the mouse cathepsin B-DARPin 4m3 complex reveals species-specific binding determinantsZarić, Miki (Avtor) Tušar, Livija (Avtor) Kramer, Lovro (Avtor) Vasiljeva, Olga (Avtor) Novak, Matej (Avtor) Impens, Francis (Avtor) Usenik, Aleksandra (Avtor) Gevaert, Kris (Avtor) Turk, Dušan (Avtor) Turk, Boris (Avtor) cathepsin Bprotein inhibitorproteomicsCathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (KD = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions.MDPI20252026-01-16 09:25:41Neznano25322Švicasl© 2025 by the authors.