Multidrug recognition and pumping by bacterial multidrug transporter,~ A structural view.
MURAKAMI, SATOSHI.
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, JapanThe emergence of bacterial multidrug resistance is anincreasing problem in the treatment of such infectious diseases.
The major cause for the multi-drug resistance of bacteria is amulti-drug efflux transporter, which exports drugs out of thecells. AcrB and its homologues are the major multi-drug efflux
transporter in gram-negative bacteria, which confer intrinsicdrug tolerance and multi-drug resistance when they areoverproduced. AcrB exports a wide variety of antibiotics,
antiseptics, anti-cancer chemotherapeutics and toxic
compounds including anionic, cationic, zwitterionic, andneutral compounds directly out of the cells bypassing theperiplasm driven by proton-motive force. It cooperates withmembrane fusion protein AcrA and outer membrane channel
Crystal structure of AcrB-drug
TolC. The X-ray crystal structure of AcrB was first solved bycomplex. (Top View)
our group in 2002 [1]. It is the first structure of not only amulti-drug efflux transporter but also a secondary active transporter, driven by proton-motive force.
In 2006, we solve the crystal structures of AcrB with and without substrates in the new crystalform [2]. The crystal used in this study has lower crystallographic symmetry than that of crystal,
which used in our previous study. The new crystal structure solved with new crystal form isasymmetric. The AcrB-drug complex consists of asymmetric three protomers, each of which hasdifferent conformation corresponding to one of the three functional states of the transport cycle. They
are “access” in which substrate incorporates, “binding” to which substrate binds, and “extrusion” bywhich substrate extrudes. Bound substrate was found in the periplasmic domain of “binding”
protomer. In the periplasmic part of each protomer, there is a substrate-binding pocket. In this pocket,
there are many aromatic amino acid residues for binding of hydrophobic substrates by aromaticaromatic interactions. Different side chains in this pocket form different binding sites to recognize
different type of substrate. The mechanism, the multi-site binding, for multiple substrate recognitionwas also found in the soluble multi-drug binding transcription factor. The pocket is expanded in the“binding” state and allows substrate binding in this voluminous pocket. The expansion of binding
pocket creates a possible exit between sub-domains forming the pocket. However, one functionally
important alfa-helix belonging to the “extrusion” protomer inclines and fills this exit to make thepocket closed. At the same time, this inclination creates an opened space from the shrunk binding
pocket in the “extrusion” protomer. So, this situation corresponds to just after extrusion of substrates
from the pocket to the exit of the AcrB, which connects to the outer membrane channel, TolC. The
remaining protomer “access” also has a shrunken binding pocket, but opened “vestibule” exposed tothe periplasmic space. Substrates are incorporated from opened vestibule in the “access” state, andbind to the different locations in the voluminous aromatic pocket in the “binding” state. Then, in the“extrusion” state, the vestibule is closed and the exit is opened. At this state, the bound substrate issqueezed out into the TolC channel by shrinking the pocket. All these structural changes are coupled
to proton translocation across the membrane. The protonation in the “extrusion” state anddeprotonation in the “access” state of three functionally important charged residues (Asp407, Asp408
and Lys940) in the transmembrane domains would affect the accessibility or influence binding orextrusion of substrate that occurring in the periplasmic domains.

Based on three different conformations and transport states observed for the three protomers of AcrB,
we propose that drugs are exported by a three-step functionally rotating mechanism in which drugs
undergo ordered binding change [2]. Such an ordered binding change mechanism in trimer is similarin principal to the mechanism of the pseudo-trimeric F1ATPase, except that AcrB has no central stalkthat undergoes mechanical rotation.

Schematic illustration of the proposed“Functionally rotating ordered multidrug binding change mechanism” mediated by AcrB.

[1] S. Murakami, R. Nakashima, E. Yamashita, and A. Yamaguchi. “Crystal structure of bacterial multidrug efflux
transporter AcrB.” Nature, 419, 587-593 (2002).
[2] S. Murakami, R. Nakashima, E. Yamashita, T. Matsumoto, and A. Yamaguchi. “Crystal structures of a multidrug
transporter reveal a functionally rotating mechanism.” Nature, 443, 173-179 (2006).