Functional Analysis of Multidrug Efflux
Transporter AcrB by All-Atom Molecular
Dynamics Simulation
Graduate School of Nanobioscience, Yokohama City University
Tsutomu Yamane (left photo)
Mitsunori Ikeguchi (right photo)
(Molecular Scale WG)
Drug resistance is a phenomenon where drugs become ineffective against pathogens or cancer cells, which leads to a major social problem. There are some known mechanisms for drug resistance. One of the causes is where membrane proteins, located in the cell membrane of the bacteria and called multidrug efflux transporters, play an important role. A multidrug efflux transporter is a membrane protein which works to discharge various cytotoxic chemicals actively to the outside of the cell.
Among RND-type multidrug efflux transporters typically found in multidrug resistant Pseudomonas aeruginosa which is the main cause of nosocomial infection, the atomic-level crystalline structure of Escherichia coli-derived AcrB was determined in 2006 by Dr. Satoshi Murakami (presently professor of Tokyo Institute of Technology), et al. According to the results, AcrB functions as an assembly of three giant proteins (homotrimer), each of which consists of approximately 1,000 amino acids. The respective proteins have three different 3-D structures that act for access, binding and extrusion of the drug, respectively (Fig. 1A). With the drive force obtained by transfer of protons into cells using the difference in proton concentration (pH) of periplasm and cytoplasm, each protein switches between the 3-D structures in turn to perform the drug-efflux function (functionally rotating mechanism, Fig. 1B). In addition, three charged amino acids located at the membrane spanning region (Asp407, Asp408, Lys940) contribute to proton transfer into cells, and the side chain structure of Lys940 is different only in the extrusion state (Fig. 1C). Through this, a change in the protonated state of these charged amino acids due to proton transfer is believed to trigger the functionally rotating mechanism.
AcrB is the common res earch t arget of our Molecular Scale Team, and its molecular simulation has been performed by various methods. Since a more realistic environment is considered in the allatom molecular dynamics simulation we use, our computation covers a system including membrane lipids, water molecules, ions and even hydrogen atoms (Fig. 2, center). Therefore, our computations have to be performed on a very large system of which the total particle number is approximately 470,000, but we can take detailed views of the various interactions in the system. Such an all-atom molecular dynamics simulation revealed the following.
(1) |
When Asp407 and Asp408 were both in the deprotonated state in the extrusion protomer, it was found that the side chain of Lys940 changes to have the structure found in the binding state and the access state (Fig. 2, right panel). Meanwhile, when only Asp408 is in the protonated state, the extrusion-type conformation was found to be stably maintained. |
(2) |
In the simulation where both Asp407 and Asp408 of the extrusion protomer were deprotonated concurrently to change the struc ture of the Lys940 side chain, the drug entry point which is usually closed in the extrusion state opens, and a structural change toward the access state was observed (Fig. 2, left panel). |
Currently we are studying to reveal the mechanisms of how a structural change of the extrusion-type Lys940 side chain leads to a structural change for opening the drug entry point.
 |
Fig.1 :Structure and drug efflux mechanism of multidrug efflux transporter AcrB |
 |
Fig.2 : System used for all-atom molecular simulation (center: a water molecule is indicated by a gray sphere, and a lipid by a blue sphere in the figure), and the results (left and right panels) |
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