A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps.

Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower alpha-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal alpha-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state.