Identification of Oligomerization and Drug-binding Domains of the Membrane Fusion Protein EmrA

• Published in: The Journal of biological chemistry 2003-04, Vol.278 (15), p.12903-12912
• Main Authors: Borges-Walmsley, M Ines, Beauchamp, Jeremy, Kelly, Sharon M, Jumel, Kornelia, Candlish, Denise, Harding, Stephen E, Price, Nicholas C, Walmsley, Adrian R
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone - pharmacology; Cloning, Molecular; Dimerization; DNA Primers; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Haemophilus influenzae - metabolism; Ion Pumps - chemistry; Ion Pumps - metabolism; Kinetics; Macromolecular Substances; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Models, Molecular; Polymerase Chain Reaction; Protein Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M209457200

Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single α-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a β-sheet subdomain and an α-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The “free” leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The β-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP.