PDBsum entry 2v4d

Membrane protein

PDB id: 2v4d

Contents

Protein chains

232 a.a.

(+ 5 more) 327 a.a.

Ligands

SO4 ×16

References listed in PDB file

Key reference

Title

The assembled structure of a complete tripartite bacterial multidrug efflux pump.

Authors

M.F.Symmons, E.Bokma, E.Koronakis, C.Hughes, V.Koronakis.

Ref.

Proc Natl Acad Sci U S A, 2009, 106, 7173-7178. [DOI no: 10.1073/pnas.0900693106]

PubMed id

19342493

Abstract

Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a beta-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous beta-roll, beta-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter-adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin-TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA(3)-AcrB(3)-TolC(3) model presents a 610,000-Da, 270-A-long efflux pump crossing the entire bacterial cell envelope.

Figure 2.
The completed structure of the periplasmic adaptor. (A) Structure of MexA including the MP domain. The 4 adaptor domains are: α-hairpin (blue), lipoyl (green), β-barrel (yellow), and MP β-roll (orange). Turns are gray except for 2 MP domain helical turns (yellow) that include Gly residues (white Cα atoms) on the concave surface effecting crystal contacts. The enlarged inset gives a smoothed representation of the MP domain topology, with elements numbered according to the adaptor family sequence alignment (Fig. S1) and colored from blue to red. Trp-309 is shown in gray. (B) The MP domain: conformational variation, rotation, and crystal contacts. The MexA adaptor barrel (yellow) and MP (orange) domain, shown in the unrotated MP domain conformation, establish crystal contacts with a neighboring copy (gray), with the MP domain in its rotated conformation. Helical turns on the MP domain concave face are in yellow, and Gly-281 and Trp-309 are shown as white Cα atoms and gray side-chains, respectively (labeled in italics on the rotated domain).

Figure 4.
Docking of AcrA and AcrB directed by the cross-linking data. (A) AcrA and AcrB interaction hotspots predocking. Cross-linking hotspots on the AcrA and AcrB surfaces predocking, colored on a gradient reflecting all cross-linking data: from dark blue near negative Cys-substituted residues (no cross-link), through green near residues with a bias to the L linker, to yellow/orange/red with increasing proximity to positives linked by both S and L linker. The TolC interface of the adaptor hairpin (23) is in magenta. The arrow indicates rotation from side to front view. (B) Docked complex of AcrA on an AcrB subunit. The 4 AcrA adaptor domains are shown in green shades, with 2 helical turns of the MP concave surface in yellow, β-turn Gly residues in white and the N-terminal residue in blue. The transporter periplasmic subdomain colors are as in Fig. 3B. Cross-sections at 2 levels of the complex (indicated by the brackets) are boxed on the right and illustrate the domain–domain contacts of the adaptor MP domain (i) and β-barrel and lipoyl domains (ii).