Graduate School of Science, Kyoto University
Shoji Takada, Xin-Qiu Yao, and Hiroo Kenzaki

Multidrug resistance where most drugs become ineffective leads to serious social problems in nosocomial infection and cancer chemotherapy, etc. This multidrug resistance arises due to different mechanisms. In the case of Pseudomonas aeruginosa which was a large problem in nosocomial infection, the main cause of multidrug resistance was that expression of a multidrug discharging transporter of RND type in Pseudomonas aeruginosa increases and discharges antibiotics from the bacteria. The multidrug discharging transporter of RND type is driven by transfer of H+ (proton) using the difference in acidity (pH) inside and outside cells, and discharges the drug by using this ability. The atomic structure of the Escherichia coli-derived RND type multidrug discharging transporter “AcrB” was elucidated by Mr. Satoshi Murakami (presently Professor of Tokyo Institute of Technology), et al. using X-ray crystal structural analysis in 2002 and 2006. In the structural analysis done in 2002, it was shown that AcrB is a trimer of 3 similar molecules, having three-fold symmetry, while in the structural analysis in 2006, it was found that each molecule has the function of a membrane proton transporter and drug discharger, and that the trimer of AcrB has an asymmetric structure. In the first molecule of this asymmetric AcrB trimer structure, a route considered as an entrance for the drug facing into the cell opens (incorporation type), in the second molecule, the drug binds to the center (binding type), and in the third molecule, a drug discharge port facing outward from the cell opens (discharging type). Murakami et al. considered that the 3 molecules of the AcrB trimer discharge drugs by mediating these 3 functional states in turn. Since it seems that the whole structure rotates 120 degrees by changing the respective states of the 3 molecules step by step, this mechanism of drug discharge was named a “functional rotation mechanism.” However, since the verification experiment using this experimental system was difficult, it was impossible to verify this hypothesis.

We have independently developed a technique for coarse graining molecular simulation of biomolecules as part of the project “Research and Development of Next-Generation Living Matter Integrated Simulation Software” of the Ministry of Education, Culture, Sports, Science and Technology. In this research, we conducted a functional simulation attributable to fluctuation of the multidrug discharge transporter AcrB by applying this new technique.

(1) Functional rotation of AcrB trimer and drug discharge: In the asymmetric AcrB trimer structure, when a proton binds to the drug-bound AcrB molecule (blue on the left side of Figure 1) from the extracellular space, the drug is discharged outward (center of Figure 1), and subsequently, the other 2 AcrB molecules also changed their state, and a functional rotation occurred (right side of Figure 1). By this, it was shown that functional rotation occurred according to proton binding and that drug discharge could occur.

(2) Resting state of AcrB trimer: It was found that, if a drug is removed from the asymmetric AcrB trimer structure, the structure having three-fold symmetry becomes stable. This structure is near the structure elucidated in 2002. That is, the structure obtained in the structural analysis in 2006 is a snapshot of the way the AcrB trimer discharges the drug, and the structure in 2002 is considered to correspond to the resting state.

Reference
Xin-Qiu Yao, Hiroo Kenzaki, Satoshi Murakami & Shoji Takada, Drug export and allosteric coupling in a multidrug transpor ter revealed by molecular simulations “Nature Communications. 1, 117 (8 pages) (2010)

Figure 1 : Drug discharge and functional rotation of AcrB due to proton binding The AcrB trimer is of asymmetric structure. In the first molecule, the route considered as an entrance for the drug, which faces into the cell, opens (green in the left figure: incorporation type), in the second molecule, the drug binds to the center (blue in the left figure: binding type), and in the third molecule, the drug discharge port facing outward opens (red in the left figure: discharging type). When a proton binds to the second drug-binding molecule from the outside of a cell (arrow of dotted line in the left figure), the drug of this molecule is discharged to the outside of the cell (figure at the center), and the other 2 molecules change their states (right figure).