9S60

Structural and proteomics analysis of the mouse cathepsin B - DARPin 4m3 complex reveals determinants of species - specific binding

This is a non-PDB format compatible entry.

Summary for 9S60

• Entry DOI: 10.2210/pdb9s60/pdb
• Descriptor: Cathepsin B, DARPin 4m3 (3 entities in total)
• Functional Keywords: cysteine cathepsin b, mechanism of inhibition, species-specific binding, darpin 4m3, hydrolase
• Biological source: Mus musculus (house mouse); More
• Total number of polymer chains: 4
• Total formula weight: 92142.12

Authors

Tusar, L.,Zaric, M.,Usenik, A.,Vasiljeva, O.,Novak, M.,Turk, D.,Turk, B. (deposition date: 2025-07-30, release date: 2026-01-14)

Primary citation

Zaric, M.,Tusar, L.,Kramer, L.,Vasiljeva, O.,Novak, M.,Impens, F.,Usenik, A.,Gevaert, K.,Turk, D.,Turk, B.
Structural and Proteomic Analysis of the Mouse Cathepsin B-DARPin 4m3 Complex Reveals Species-Specific Binding Determinants.
Int J Mol Sci, 26:-, 2025

PubMed Abstract: Cathepsin 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 (K = 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.

PubMed: 41465336
DOI: 10.3390/ijms262411910

Experimental method

X-RAY DIFFRACTION (1.69 Å)