Water Pathway Analysis of Multi-Drug Efflux Transporter AcrB

Abstract

The multi-drug efflux transporter AcrB exists in the inner membrane region of E. coli., and exports wide variety of noxious compounds using proton motive force as an energy source in Gram-negative bacteria. From the results of x-ray crystallography, the followings were found: (1) AcrB adopts asymmetric structure comprising three protomers with different structures, which correspond to access (A), binding (B) and extrusion (E) state of drugs, (2) three titratable residues (Asp407, Asp408 and Lys940), which locate in the middle of the transmembrane domain, form the protonation site (PS), and the Lys940 in E state adopts the different conformation from those of other states. These results suggest that AcrB exports drugs through the cyclic structural change among three states by using proton motive force generated by the change of protonation state in PS, which are called “functional rotation”.We have studied the functional rotation mechanism of AcrB by using the Motion-tree method, which is a new procedure to describe the structural change as rigid body motions with a hierarchal manner. Our results have elucidated how convert the proton motive force generated from PS to the large structural change of AcrB.In the present study, to clarify the proton transfer process in the transmembrane domain, we searched the water pathways from the PS by using CAVER and 3D-RISM methods.As the results from CAVER method, A and B state protomers observed pathways from the PS to the periplasm side, and E state protomer observed a pathway from PS to the cytoplasm side. In addition, the results of 3D-RISM method suggest that B and E state pathways have an ability to permeate water molecules. These results were consistent with the protonation states of each protomers estimated from structural change process from Motion-tree method.