Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine.

Ming Liu,Jie Heng,Xianping Wang,Yuan Gao

doi:10.1007/s41048-016-0028-1

Abstract

The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family. Two crystal structures were produced for E. coli MdfA, one in complex with acetylcholine and the other with potential reserpine, which are substrate and inhibitor of VNTs, respectively. These structures show that the binding sites of these two molecules are different. The Ach-binding MfdA is mainly dependent on D34, while reserpine-binding site is more hydrophobic. Based on sequence alignment and homology modelling, we were able to provide mechanistic insights into the association between the inhibition and the conformational changes of these transporters.Electronic supplementary materialThe online version of this article (doi:10.1007/s41048-016-0028-1) contains supplementary material, which is available to authorized users.

Highlights

MdfA, as a typical antiporter of the major facilitator superfamily (MFS), has been the subject of extensive study, especially in the research of multidrug transport mechanisms (Edgar and Bibi 1997)
We found that three acidic residues of vesicular acetylcholine transporter (VAChT), D33, E309 and D398, correspond with residues E26, I239 and N331 of E. coli MdfA (ecMdfA), respectively, which are located near the ACh-binding pocket
According to the multiple sequence alignment of the 12 transmembrane proton-dependent bacterial multidrug transporters of the MFS family, these transporters contain a number of highly conserved motifs, which indicates that they may share a similar transportation mechanism (Putman et al 2000)

Summary

Introduction

MdfA, as a typical antiporter of the major facilitator superfamily (MFS), has been the subject of extensive study, especially in the research of multidrug transport mechanisms (Edgar and Bibi 1997). Crystal structures of E. coli MdfA (ecMdfA) have recently been reported by our laboratory (Heng et al 2015). Based on the parsed structures of ecMdfA, the key location for the binding of a variety of substrates was determined to be the large. D34, is present deep within this cavity, and is proposed to be critical for the binding of certain substrates (Heng et al 2015). The substrate-bound crystal structure of MdfA provided a base for further structural and functional study of other homologous proteins, such as MdtM, another MFS transporter, which share high sequence identity with MdfA (Paul et al 2014)

Methods

Results

Conclusion