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Background  Cathepsin B (catB) is a promising target for anti-cancer drug design due to its implication in several steps of tumorigenesis. catB activity and inhibition are pH-dependent, making it difficult to identify efficient inhibitor candidates for clinical trials. In addition it is known that heparin binding stabilizes the enzyme in alkaline conditions. However, the molecular mechanism of stabilization is not well understood, indicating the need for more detailed structural and dynamic studies in order to clarify the influence of pH and heparin binding on catB stability.    Results  Our pKa calculations of catB titratable residues revealed distinct protonation states under different pH conditions for six key residues, of which four lie in the crucial interdomain interface. This implies changes in the overall charge distribution at the catB surface, as revealed by calculation of the electrostatic potential. We identified two basic surface regions as possible heparin binding sites, which were confirmed by docking calculations. Molecular dynamics (MD) of both  apo  catB and catB-heparin complexes were performed using protonation states for catB residues corresponding to the relevant acidic or alkaline conditions. The MD of  apo  catB at pH 5.5 was very stable, and presented the highest number and occupancy of hydrogen bonds within the inter-domain interface. In contrast, under alkaline conditions the enzyme's overall flexibility was increased: interactions between active site residues were lost, helical content decreased, and domain separation was observed as well as high-amplitude motions of the occluding loop – a main target of drug design studies. Essential dynamics analysis revealed that heparin binding modulates large amplitude motions promoting rearrangement of contacts between catB domains, thus favoring the maintenance of helical content as well as active site stability.    Conclusions  The results of our study contribute to unraveling the molecular events involved in catB inactivation in alkaline pH, highlighting the fact that protonation changes of few residues can alter the overall dynamics of an enzyme. Moreover, we propose an allosteric role for heparin in the regulation of catB stability in such a manner that the restriction of enzyme flexibility would allow the establishment of stronger contacts and thus the maintenance of overall structure.

How does heparin prevent the pH inactivation of cathepsin B? Allosteric mechanism elucidated by docking and molecular dynamics