Abstract

Cathepsins are predominantly cysteine proteases that function in lysosomes and the extracellular matrix, where they regulate essential proteolytic processes. They are synthesised as inactive zymogens (procathepsins), in which an N-terminal propeptide blocks access to the active site and is removed during maturation. Glycosaminoglycans (GAGs), a class of linear, sulfated polysaccharides composed of repeating disaccharide units, are known to modulate both cathepsin activity and proenzyme processing. Here, we use molecular modelling to elucidate the role of GAGs in the maturation of procathepsin K. Molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations identify putative allosteric sites on the procathepsin surface that mediate GAG recognition. Microsecond-scale MD simulations of the most stable complexes, analysed via principal component analysis (PCA), reveal GAG-dependent shifts in the conformational landscape of the proenzyme. To probe environmental effects, we further simulate apo and GAG-bound procathepsin K under lysosomal conditions (pH 4). These simulations demonstrate a synergistic interplay between acidification and GAG binding in promoting activation. While low pH destabilises propeptide secondary structure, GAG binding at a distinct allosteric hotspot amplifies this effect, promoting helix unwinding and facilitating propeptide dissociation. Overall, our results provide a molecular-level framework for GAG-assisted maturation of procathepsin K, highlighting cooperative environmental and allosteric regulation of zymogen activation.