Connecting the dots in the mechanism of action of Cucurbitacin E (CurE) – path analysis and steered molecular dynamics reveal the precise site of entry and the passage of CurE in filamentous actin

Cucurbitacin E (CurE) modulates actin cytoskeleton by forming an irreversible covalent bond with Cys257 of actin. The reported binding conformation of CurE is deeply buried in the subdomain 4 of actin and is closely situated to the ATP-binding site. The entry and the path taken by CurE to reach this buried site remain a mystery. In this study, steered molecular dynamics (SMD) simulations were conducted to delineate the diffusion of CurE to its binding site. SMD simulations reveal that the distinctive entry site of CurE found in subdomain 4 is by itself a closed and compact region of two loops lying beside each other like a closed door and CurE induces it to open. From this point, CurE moves toward its binding site through a path facilitated by Thr188, Leu261, Ile267, Ile309, Tyr306. This study is also an insight into how CurE distinctly differentiates its tunnel to the binding site from the ATP-binding site. The conformational changes of CurE along the path to the binding site are surprisingly very minimal and closely resemble the attack conformation at the end of simulation. The study reveals that the little energy spent by the molecule is compensated by the enthalpic contribution to binding-free energy barrier making it undoubtedly the most preferred path of CurE. This study is the first of its kind in which the SMD was used to derive the complete and continuous translocation of one of the most potent phytochemicals, CurE through the binding site gorge of actin. Communicated by Ramaswamy H. Sarma