The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC.
Efflux pumps play a major role in multidrug resistance of pathogenic bacteria. The TolC homologue HI1462 was identified as the single channel-tunnel in Haemophilus influenzae required to form a functional multidrug efflux pump. The outer-membrane protein was expressed in Escherichia coli, purified and reconstituted in black lipid membranes. It exhibited a comparatively small single-channel conductance of 43 pS in 1 M KCl and is the first known TolC homologue which is anion-selective. The HI1462 structure was modelled and an arginine residue lining the tunnel entrance was identified. The channel-tunnel of a mutant with the arginine substituted by an alanine residue was cation-selective and had a sevenfold higher single-channel conductance compared to wild-type. These results confirm that the arginine is responsible for anion selectivity and forms a salt bridge with a glutamate residue of the adjacent monomer, establishing a circular network, which keeps the tunnel entrance in a tightly closed conformation. In in vivo experiments, both the wild-type HI1462 and the mutant were able to substitute for E. coli TolC in the haemolysin secretion system, but not in the AcrAB/TolC multidrug efflux pump. The structure-function relationship of HI1462 is discussed in the context of the well-studied TolC channel-tunnel of E. coli.
Polleichtner
Georg
G
Universität Würzburg, Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Andersen
Christian
C
Universität Würzburg, Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006193:Haemophilus influenzae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006460:Hemolysin Proteins / Q000378:metabolism; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli