X-ray structure of EmrE supports dual topology model

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.One mechanism of multidrug resistance involves the active extrusion of toxic molecules out of the cell by dedicated membrane transporters. In prokaryotes, five superfamilies of transporters handle vectorial drug traffic moving energetically uphill against substrate concentration gradients (1). The thermodynamics of the problem are rendered favorable through coupling of substrate movement to the direct use of ATP energy or the discharge of electrochemical ion gradients. Small multidrug resistance transporters are the smallest bacterial transporters with four predicted transmembrane ␣-helices and no significant extramembrane domain (2, 3). They function as dimers coupling translocation of positively charged hydrophobic substrates out of the cell to the inward movement of protons.Much of the current mechanistic understanding of small multidrug resistance transporters has emerged from seminal studies in the laboratory of Schuldiner and co-workers (4 -7)defining fundamental steps in the transport cycle of Escherichia coli EmrE. Protons and substrates share a common binding site organized around the absolutely conserved, membrane-embedded, glutamate 14 in transmembrane (TM) 3 helix 1 (8). Binding of positively charged hydrophobic substrates is coordinated by the Glu-14 side chain and stabilized by interactions with aromatic res...

Section: Resultssupporting

confidence: 92%

Section: Discussionmentioning

confidence: 89%

mentioning

confidence: 83%

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Structure, Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

Amadi¹,

Koteiche²,

Mishra

et al. 2010

“…The claim for an antiparallel topology was supported by a reinterpretation of the electron density maps of two-dimensional crystals of EmrE that showed that parts of the structure are related by quasisymmetry (37). A C␣ model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix (34) and was supported by a re-evaluation of the data published in the retracted papers (38). Our own work demonstrates that dimers with parallel topology are functional and the functionality of antiparallel dimers remains to be established (36,39,40).…”

Section: Discussionmentioning

confidence: 95%

Identification of a Glycine Motif Required for Packing in EmrE, a Multidrug Transporter from Escherichia coli

Elbaz

Salomon

Schuldiner

2008

Glycine residues may play functional and structural roles in membrane proteins. In this work we studied the role of glycine residues in EmrE, a small multidrug transporter from Escherichia coli. EmrE extrudes various drugs across the plasma membrane in exchange with protons and, as a result, confers resistance against their toxic effects. Each of 12 glycine residues was replaced by site-directed mutagenesis. Four of the 12 glycine residues in EmrE are evolutionary conserved within the small multidrug resistance family of multidrug transporters. Our analysis reveals that only two (Gly-67 and Gly-97) of these four highly conserved residues are essential for transporter activity. Moreover, two glycine positions that are less conserved, Gly-17 and Gly-90, demonstrate also a nil phenotype when substituted. Our present results identifying Gly-17 and Gly-67 as irreplaceable reinforce the importance of previously defined functional clusters. Two essential glycine residues, Gly-90 and Gly-97, form a protein motif in which glycine residues are separated by six other residues (GG7). Upon substitution of glycine in these positions, the protein ability to form dimers is impaired as evaluated by cross-linking and pull-down experiments.

“…Proteins with dual topology are known to have weak signals (net differences in charged amino acids next to membrane boundaries involved in determining membrane topology) adjacent to transmembrane domains (30). Interestingly, for at least one known bacterial protein, the relative proportions of the opposing topological orientations depend on growth conditions (10,33), raising the possibilities that (i) the ratio between the two orientations may not always be unity, and (ii) post/co-translational modification(s) may alter the net charges at critical membrane boundary regions of the protein sequence so as to control the relative proportions of the two alternative orientations depending on the prevailing activity of protein modifying enzymes. If these considerations can be extrapolated to DGAT1, they may explain our previous observations that the relative amounts of overt and latent DGAT activity in the ER of rat liver (which will be dependent on the relative distribution of DGAT1 activity as this accounts for the majority of the overt DGAT activity) are capable of modulation by physiological state and by hypolipidemic drug treatment (6).…”

Section: Discussionmentioning

confidence: 99%

Evidence That Diacylglycerol Acyltransferase 1 (DGAT1) Has Dual Membrane Topology in the Endoplasmic Reticulum of HepG2 Cells

Wurie

Buckett

Zammit

2011

Triacylglycerol (TAG) synthesis and secretion are important functions of the liver that have major impacts on health, as overaccumulation of TAG within the liver (steatosis) or hypersecretion of TAG within very low density lipoproteins (VLDL) both have deleterious metabolic consequences. Two diacylglycerol acyltransferases (DGATs 1 and 2) can catalyze the final step in the synthesis of TAG from diacylglycerol, which has been suggested to play an important role in the transfer of the glyceride moiety across the endoplasmic reticular membrane for (re)synthesis of TAG on the lumenal aspect of the endoplasmic reticular (ER) membrane (Owen, M., Corstorphine, C. C., and Zammit, V. A. (1997) Biochem. J. 323, 17-21). Recent topographical studies suggested that the oligomeric enzyme DGAT1 is exclusively lumen facing (latent) in the ER membrane. By contrast, in the present study, using two specific inhibitors of human DGAT1, we present evidence that DGAT1 has a dual topology within the ER of HepG2 cells, with approximately equal DGAT1 activities exposed on the cytosolic and lumenal aspects of the ER membrane. This was confirmed by the observation of the loss of both overt (partial) and latent (total) DGAT activity in microsomes prepared from livers of Dgat1 ؊/؊ mice. Conformational differences between DGAT1 molecules having the different topologies were indicated by the markedly disparate sensitivities of the overt DGAT1 to one of the inhibitors. These data suggest that DGAT1 belongs to the family of oligomeric membrane proteins that adopt a dual membrane topology.Hypertriglyceridemia is a key biomarker for the metabolic/ insulin resistance syndrome and for associated morbidities, including type-2 diabetes and cardiovascular disease (1). Similarly, excessive accumulation of triglycerides in cytoplasmic lipid droplets results in hepatic steatosis, now recognized as being associated, possibly causatively, with whole body insulin resistance, and which may progress to nonalcoholic fatty liver or steatohepatitis (2, 3). Fasting hypertriglyceridemia is primarily due to the hypersecretion of triglyceride (TAG) 3 by the liver, within very low density lipoproteins (VLDL). Therefore, an understanding of the enzymology involved in triglyceride synthesis, remodeling, storage, and assembly into secreted VLDL is essential for the design of pharmacological strategies aimed at managing dyslipidaemia without the exacerbation of hepatic steatosis, and vice versa.Diacylglycerol acyltransferases (DGATs) catalyze the final reaction of TAG synthesis. Two distinct gene products, DGAT1 and DGAT2, that catalyze most of tissue TAG synthesis have been described (4, 5) but remain relatively poorly characterized, and their respective, nonredundant functions are still to be elucidated. In the present study, we have used two specific inhibitors (which belong to different chemical classes of compounds) of human DGAT1, in combination with the selective permeabilization of the plasma membrane and the ER membrane of whole hepatocytes, to study the sidednes...