The CusCFBA metal efflux pump of Escherichia Coli serves to protect the bacteria in environments with lethal concentrations of Ag(I) and Cu(I). Its periplasmic piece, the membrane fusion protein CusB, has an extremely disordered N-terminus, and therefore could not be resolved by crystallography. This region is very important both structurally and functionally because it has been experimentally proven to transfer the metal by itself from the metallochaperone CusF and to induce a structural change in the rest of CusB to increase Cu(I)/Ag(I) resistance. The structural and dynamic characterization of the N-terminal region of CusB lays out a crucial step to understand the metal uptake by the entire CusCFBA pump in periplasm because this region contains the metal binding site. We ran extensive molecular dynamics simulations on both apo and Cu(I)-bound versions of it and observed that the metal binding causes only a slight, local stabilization around the metal binding site. Most of the disorder was traced back to the tails of the chain and we suggest that dismissing the tails would not affect the functional competency of this protein domain as a metal chelator. To gain more insight into the metal transfer between CusF and CusB, we docked the recently discovered open flap structure of CusF to metal-bound CusB N-terminus and simulated two viable metal sites involving both of these proteins as potential steps of the transfer process. The N-terminus of CusB became much more ordered. The metal site dominated by CusB residues showed an increased stability in comparison to that dominated by CusF residues. Given the experimental finding of similar metal binding affinities of these two proteins, this might play a role in determining the direction of the metal transfer: from CusF to CusB which is towards the exterior of the cell.

Insights into the structure and dynamics of the Cu(I) binding proteins CusB and CusF

247th National Meeting (2014)

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