The Structure and Dynamics of EmrE in Liposomes

Abstract

Site-directed spin labeling and electron paramagnetic resonance were used to investigate the structure and conformational dynamics of the small multidrug resistance transporter EmrE from Escherichia coli. The data set consists of the mobilities, solvent accessibilities of 110 spin labeled residues as well as selected pairwise distances. These parameters were interpreted as constraints on the local steric environment, the orientation of helices in the lipid phase and packing of the transmembrane helices in each monomer as well as across the dimer interface. The data collectively suggest that in liposomes, ligand-free EmrE average structure is not compatible with the crystal structure and an EM-based model. Spin label pairs at the dimer interface located at opposite sides of helix 3 reveal dipolar interaction between the spins separated by less than 15A which contradicts their inverted relative orientation in the crystal structure. Furthermore, distances between spin labels along helix 4 are not consistent with its tight packing at the dimer interface. The spectroscopic data is supported by an expanded topological analysis of EmrE-GFP chimeras which reveals that the previous interpretation of Rapp et al is not unequivocal. Binding of tetraphenyl phosphonium (TPP+) to EmrE increases the structural order as manifested by a narrowing of spin label distance distributions but does not lead to major conformational rearrangements. The EPR-based constraints are being used to generate a model of EmrE in liposomes.