Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC

Journal of Molecular Biology
Matthew K HigginsVassilis Koronakis

Abstract

The trimeric TolC protein of Escherichia coli comprises an outer membrane beta-barrel and a contiguous alpha-helical barrel projecting across the periplasm. This provides a single 140 A long pore for multidrug efflux and protein export. We have previously reported that trivalent cations such as hexammine cobalt can severely inhibit the conductivity of the TolC pore reconstituted in planar lipid bilayers. Here, isothermal calorimetry shows that Co(NH(3))(6)(3+) binds to TolC with an affinity of 20 nM. The crystal structure of the TolC-Co(NH(3))(6)(3+) complex was determined to 2.75 A resolution, and showed no significant difference in the protein when compared with unliganded TolC. An electron density difference map revealed that a single ligand molecule binds at the centre of the periplasmic entrance, the sole constriction of TolC. The octahedral symmetry of the ligand and the three-fold rotational symmetry of the TolC entrance determine a binding site in which the ligand forms hydrogen bonds with the Asp(374) residue of each monomer. When Asp(374) was substituted by alanine, high affinity ligand binding was abolished and inhibition of TolC pore conductivity in lipid bilayers was alleviated. Comparable effects followed independ...Continue Reading

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Citations

Oct 8, 2013·The Protein Journal·Yee Siew ChoongTheam Soon Lim
Feb 9, 2012·Biochemistry·Martin Raunest, Christian Kandt
Aug 4, 2006·Journal of Bacteriology·David S H KimMilton H Saier
Jul 3, 2013·Annual Review of Microbiology·Philip HinchliffeVassilis Koronakis
Jan 28, 2014·Interdisciplinary Sciences, Computational Life Sciences·Nadine FischerChristian Kandt
Mar 5, 2011·Journal of Molecular Graphics & Modelling·Yee Siew ChoongAsma Ismail
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Jun 28, 2021·Molecular Biology and Evolution·Yusuf Talha TamerErdal Toprak

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