EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a ''dual topology'' model. membrane protein structure ͉ multidrug transport ͉ SMR family O ne of the mechanisms by which cells neutralize the effect of toxic compounds is through the action of membrane transporters. Secondary transporters, such as the EmrE protein of Escherichia coli, couple the efflux of drugs to the inward movement of protons across the cell membrane (refs. 1 and 2, and references therein). EmrE is the prototypical member of the SMR (small multidrug resistance) family and is one of the smallest known transporters in nature, composed of only 110 amino acid residues. Studies have shown that the basic functional unit of EmrE is an oligomer, as would be expected for a membrane protein of its small size. It appears established that the basic functional unit of EmrE is a homodimer, as shown by oligomerization assays, substrate binding experiments, negative dominance studies, and crosslinking analyses (3-8). This conclusion is further supported by the existence of paired SMR proteins, such as YdgE/YdgF of E. coli, and EbrA/EbrB and YkkC/YkkD of Bacillus subtilis. These transporters require coexpression of the two component polypeptides for proper drug efflux activity and presumably form heterodimers analogous to the EmrE homodimer (9-13).Electron microscopy (EM) studies of EmrE in complex with a transport substrate, tetraphenylphosphonium (TPP), and reconstituted in lipid bilayers have revealed the overall architecture of the transporter at 7.5 Å resolution in-plane and 16 Å perpendicular to the membrane (14). EmrE binds TPP as an asymmetric dimer, in a chamber that appears open to one side of the bilayer. Each monomer is composed of four transmembrane (TM) helices. Because of the low resolution, however, the monomers could not be delineated unambiguously, nor the TM segments assigned in a sequence-specific manner based on the experimental map alone.We have previously reported x-ray crystal structures of EmrE in the unbound form and in complex with TPP (15, 16). Regrettably, the electron density maps were calculated in the wrong hand because of an unfortunate change of sign of the anomalous differences (17), and helices were misassigned in the TPP-bound model. Recalculation of the...

A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction ‘mode A’ (proteins were expressed as precipitant) and ‘mode B’ (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of ‘mode B’ and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.