A key step in biological copper trafficking is the transfer of Cu(I) ion from the copper chaperone to the metal binding domain (MBD) of a Cu-ATPase residing on the membrane of an intracellular organelle. The molecular mechanism of the transfer process constitutes sequential formation of multiple covalently linked copper mediated protein-protein complexes. NMR studies and molecular dynamics (MD) simulations revealed existence of strong electrostatic interactions in a late-stage intermediate and highly fluctuating nature of the Cu-binding loop of apo-MBD. Because of a timescale separation between protein motions and the chemical event of bond formation, several scenarios could unfold in the early stages of the transfer process. To understand these unexplored mechanistic issues, we employed MD simulation-based free-energy calculations to investigate the formation of protein-protein complexes between Cu(I)-loaded holo-chaperone and apo-MBD from yeast. Our calculations revealed a new minimum, where proteins were arranged in a more open conformation separated by solvent, along with a tightly bound complex with multiple salt bridges resembling the experimental structure. Unbiased trajectories launched from the free-energy basins, aided by QM/MM calculations, confirmed the existence of multiple reaction channels with varying degrees of inter-protein electrostatic interactions. These results suggested that the initial stages of the multi-step copper transfer were not entirely driven by electrostatic control and a significant role of protein dynamics. However, electrostatics played a crucial role. Without the minimum number of salt bridges, no ensemble with significant population was found that could lead to a productive chemical reaction. The interplay between these two effects is a manifestation of enthalpy-entropy compensation. From a functional perspective, our findings indicate that this essential biological pathway evolved to maximize the rate of transfer without optimizing the strong binding of trafficking partner proteins.
Avisek Das (Sun,) studied this question.