Analysis of Integral Membrane Inter and Intra Contacts in Model Multidrug Transporter EmrE using a Bacterial Two-Hybrid Method

Abstract

The bacterial cytoplasmic membrane small multidrug resistance family transporter EmrE consists of four transmembrane helices (TMH) connected by short loops and is considered to multimerize in order to export quaternary cationic compounds from the cytoplasm. Previous consensus on multimerization proposes a facial monomer-monomer interaction located in the C-terminal TMH 4, which is complicated by the mounting evidence of an antiparallel topology dimer and requires additional data on interactions which form dimers or potential higher multimeric states. Bacterial two hybrid method is used as an in vivo approach to protein-protein interaction to this end.In order to characterize regions of interaction between potential parallel or antiparallel dimers, chimeric constructs of full length monomers were made with N- and C-terminal adenylate cyclise domains. Constructs were also made of each TMH topologically constrained with maltose binding protein to the periplasmic side and one of two domains of adenylate cyclase for the bacterial two-hybrid system to the cytoplasmic side. These constructs were then cloned to represent the alternate topology. Complimentary constructs were co-expressed in an in vivo system where interaction strength between helices was quantified with the use of the Miller assay. This assay relies on the production of the o-nitrophenol anion by proportional production of β-galactosidase resulting from the transmembrane helix mediated interaction of the adenylate cyclase domains encoded into the chimeric constructs.Resulting data obtained using full length proteins and individual TMH in different orientations provides information on helix interactions and topology which compliment the in vitro structural information.