Proton Transport Mechanism of the E. coli Copper Transport Efflux Pump

Gram Negative Bacteria such as E. Coli use a tripartite complex system to expel toxic chemicals, such as antibiotics, and ions out of the cell. In E.Coli, CusCBA, which is a complex of an inner membrane transporter (CusA), a connecting fusion protein (CusB), and an outer membrane channel (CusC), utilizes the proton motive force to transport copper and silver ions out of the cell. Three charged residues in the transmembrane domain have been suggested to play a crucial role in the proton transport across the membrane. The crystal structures of the complex of CusA and CusB have recently been resolved in the presence and absence of the copper ions, providing the first atomic resolution images of the assembly of the transporter and the fusion proteins in the tripartite family. Using the crystal structure of the Apo-CusBA, we have studied the proton transport mechanism in a series of unbiased molecular dynamics simulations corresponding to different protonation schemes of these three residues. The simulations have revealed two separate water permeation pathways in the transmembrane domain that coincide with the three key residues involved in the proton transport process. The presence, stability, and the number of water molecules in the two canals show a strong correlation with protonation state of the three key residues. For instance, protonation of Asp405 leads to entrance of significantly higher number of water molecules into the protein, and deprotonation of Lys984 leads to a reduction in the number of water molecules. Moreover, protonation of Asp405 in the apo state resulted in conformational changes in the transmembrane region (TM8) and the peripelasmic cleft of the pump, initiating a transition toward the Cu-bound conformation of the protein.