Structure of the periplasmic component of a bacterial drug efflux pump

Abstract

Multidrug resistance among Gram-negative bacteria is conferred by three-component membrane pumps that expel diverse antibiotics from the cell. These efflux pumps consist of an inner membrane transporter such as the AcrB proton antiporter, an outer membrane exit duct of the TolC family, and a periplasmic protein known as the adaptor. We present the x-ray structure of the MexA adaptor from the human pathogen Pseudomonas aeruginosa. The elongated molecule contains three linearly arranged subdomains; a 47-Å-long ␣-helical hairpin, a lipoyl domain, and a six-stranded ␤-barrel. In the crystal, hairpins of neighboring MexA monomers pack side-by-side to form twisted arcs. We discuss the implications of the packing of molecules within the crystal. On the basis of the structure and packing, we suggest a model for the key periplasmic interaction between the outer membrane channel and the adaptor protein in the assembled drug efflux pump.

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

“…In the simplest interpretation, our results suggest a single binding site on the TolC protomer, interacting with the ␣-helical hairpin of a single adapter molecule. We saw no evidence for interaction with residues facing the TolC monomer interface on helices H3/H4/ H7* with any of the cross-linking agents, but weaker secondary contact sites cannot be excluded especially as ITC data indicate concentration-dependent binding events (6), and physically there would appear to be space to accommodate up to three adaptor subunits per TolC monomer (18,20).…”

“…A possible explanation of this is that the TolC coordinates used for the AcrA docking are those of the closed form and the observed space allows the movement of the TolC inner coiled-coil during transition to the open state. Consistent with such a key role, a number of these residues, particularly those directed toward TolC, are conserved in the AcrA protein family (18). To test this possibility, we simulated the opening in the docked complex by forcing movement of the lower region of the TolC inner coiled-coil (from residue Ala 347 to Ala 382 , Fig.…”

“…These interactions can be detected by cross-linking in vivo and measured in vitro by isothermal calorimetry (ITC) (6). Adaptor monomers have an elongated modular structure comprising a ␤-barrel, a lipoyl domain, and a long ␣-helical hairpin that extends over four or five heptad repeats and projects into the periplasm (18)(19)(20).…”

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

“…Of the 11 RND efflux pumps identified in this organism, 10 pumps, MexAB-OprM (Poole et al, 1993), MexCD-OprJ (Poole et al, 1996), MexEF-OprN (Kohler et al, 1997), MexGHI-OpmD (Aendekerk et al, 2002;Sekiya et al, 2003), MexJK (Chuanchuen et al, 2002), MexMN (Mima et al, 2005), MexPQ-OpmE (Mima et al, 2005), MexVW (Li et al, 2003), MexXY (Mine et al, 1999;Westbrock-Wadman et al, 1999) and TriABCOpmH (Mima et al, 2007), have been experimentally confirmed, and their properties have been reported. The RND-type efflux pump usually consists of three components to fulfil the function properly and includes an innermembrane component (RND component), a periplasmic component (MFP component), and an outer-membrane component (OMP component) (Touze et al, 2004), and the three-dimensional structures of MexA, MexB and OprM have been reported (Akama et al, 2004a, b;Higgins et al, 2004;Sennhauser et al, 2009). However, regarding the most recently analysed RND efflux pump in P. aeruginosa, TriABC-OpmH, it has been reported that this system needs four components: an RND component, two MFP components and an OMP component (Mima et al, 2007).…”

Trinity revealed: Stoichiometric complex assembly of a bacterial multidrug efflux pump

“…The energy source for drug transport is derived from the proton motive force and these transporters function as drug/proton antiporters. For all 3 individual components, structural information based on X-ray crystallography is available (4)(5)(6)(7)(8)(9)(10)(11) and has led to several suggestions of tripartite pump assemblies (10-13) (Fig. 1).…”