Evidence for Assembly of Small Multidrug Resistance Proteins by a “Two-faced” Transmembrane Helix

Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism. Clinically significant bacterial resistance to drugs and cytotoxic compounds can be conferred by the energy-dependent efflux of toxicants, catalyzed by proteins embedded in the bacterial cell membrane. One such group of proteins, the small multidrug resistance family, are drug/proton antiporters that must oligomerize to function, a process that requires the assembly of at least two inactive monomers by intermolecular association of their four transmembrane helices. Here, we have used peptides that correspond to each of the four wild type transmembrane helices of the Halobacterium salinarum protein Hsmr and a corresponding library of mutant peptides to determine the interactive surfaces that likely contribute to protein oligomerization. Hsmr peptides were examined for strong (sodium dodecyl sulfate-resistant) and weaker (perfluorooctanoate-resistant) helix-helix interactions, in conjunction with circular dichroism, fluorescence energy transfer measurements, and molecular modeling. The results are compatible with a scheme in which two faces of helix four permit self-assembly via a higher affinity asymmetric pairing and a lower affinity symmetric interaction, resulting in a discrete tetramer. Our finding that two surfaces of helix four can contribute to the stability of small multidrug resistance protein assembly provides a molecular basis for the design of therapeutics that target this antibiotic resistance mechanism. The advances made in controlling and treating infectious diseases using antibiotics are threatened by the increase of multidrug resistance in pathogenic organisms (1Walsh C. Nature. 2000; 406: 775-781Crossref PubMed Scopus (1199) Google Scholar). Clinically significant resistance to therapeutic compounds can be conferred by proteins embedded in the bacterial cell membrane that use energy-dependent mechanisms to extrude a wide variety of toxicants from the cell (2Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1271) Google Scholar). Among these, the small multidrug resistance (SMR) 4The abbreviations used are: SMR, small multidrug resistance; TM, transmembrane; WT, wild type; FRET, fluorescence resonance energy transfer; PFO, sodium perfluorooctanoate; TAMRA, 5- (and 6-) carboxytetramethylrhodamine; dansyl, 5-dimethyl-amino-1-naphthalenesulfamoyl; dabcyl, 4-dimethylaminoazobenzene-4′-sulfamoyl; MES, 4-morpholineethanesulfonic acid. proteins are highly prevalent (>60 homologs in both Gram-positive and Gram-negative bacteria (3Ninio S. Rotem D. Schuldiner S. J. Biol. Chem. 2001; 276: 48250-48256Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)) proton/drug antiporters of 100–110 amino acids (4Paulsen I.T. Skurray R.A. Tam R. Saier Jr., M.H. Turner R.J. Weiner J.H. Goldberg E.B. Grinius L.L. Mol. Microbiol. 1996; 19: 1167-1175Crossref PubMed Scopus (245) Google Scholar). SMRs catalyze the efflux of large (up to 10 Ä cross-section), structurally diverse hydrophobic cations such as acriflavine, ethidium, methyl viologen, tetracycline, and tetraphenylphosphonium (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar). The monomer of the best characterized family member, the Escherichia coli SMR protein EmrE, is a tightly packed bundle of four antiparallel transmembrane (TM) α-helices with short loops (6Arkin I.T. Russ W.P. Lebendiker M. Schuldiner S. Biochemistry. 1996; 35: 7233-7238Crossref PubMed Scopus (100) Google Scholar, 7Schwaiger M. Lebendiker M. Yerushalmi H. Coles M. Groger A. Schwarz C. Schuldiner S. Kessler H. Eur. J. Biochem. 1998; 254: 610-619Crossref PubMed Scopus (89) Google Scholar). The minimum structural unit of EmrE is a dimer that is unusual in that its component monomers have distinct conformations (8Tate C.G. Kunji E.R. Lebendiker M. Schuldiner S. EMBO J. 2001; 20: 77-81Crossref PubMed Scopus (103) Google Scholar, 9Ubarretxena-Belandia I. Baldwin J.M. Schuldiner S. Tate C.G. EMBO J. 2003; 22: 6175-6181Crossref PubMed Scopus (171) Google Scholar, 10Tate C.G. Ubarretxena-Belandia I. Baldwin J.M. J. Mol. Biol. 2003; 332: 229-242Crossref PubMed Scopus (71) Google Scholar, 11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar). The structural inequivalence of these subunits may relate to an antiparallel insertion of individual EmrE polypeptides (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar, 9Ubarretxena-Belandia I. Baldwin J.M. Schuldiner S. Tate C.G. EMBO J. 2003; 22: 6175-6181Crossref PubMed Scopus (171) Google Scholar, 11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 12Pornillos O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar), although evidence for antiparallel topology was not obtained in cysteine accessibility and labeling studies (13Ninio S. Elbaz Y. Schuldiner S. FEBS Lett. 2004; 562: 193-196Crossref PubMed Scopus (48) Google Scholar). It is also unresolved whether the pre-existence of alternate monomer folds is required for, or is the consequence of, multimer formation. Although the in vivo oligomeric state of EmrE may be a dimer of dimers (11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 14Elbaz Y. Steiner-Mordoch S. Danieli T. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1519-1524Crossref PubMed Scopus (125) Google Scholar), drug efflux activity requires the assembly of two SMR molecules (15Yerushalmi H. Lebendiker M. Schuldiner S. J. Biol. Chem. 1996; 271: 31044-31048Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). A detailed understanding of the TM helix-helix interactions that stabilize the self-assembly of SMR drug pumps is thus of considerable interest in the development of therapeutics that could target this resistance mechanism by disrupting SMR oligomerization. Like other multimeric membrane proteins, SMRs are stabilized by weak (van der Waals and electrostatic) intermolecular interactions between TM helix faces, defined as individual sets of residues on distinct surfaces of each α-helical TM segment (16Popot J.L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (823) Google Scholar). The intermolecular helix-helix interactions that assemble SMR oligomers vary in strength among family members (17Ninio S. Schuldiner S. J. Biol. Chem. 2003; 278: 12000-12005Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) but are of sufficient stability in the Halobacterium salinarum homolog Hsmr to resist the strongly denaturing conditions of SDS-PAGE that disrupt oligomerization of EmrE (17Ninio S. Schuldiner S. J. Biol. Chem. 2003; 278: 12000-12005Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The SDS-resistant Hsmr dimer thus allows for the detection of key SMR intermolecular interactions that could be denatured in its less stable counterparts. We have independently been studying Hsmr as a model system, using peptides that correspond to each of its four wild type (WT) TM helices, along with a corresponding library of mutant peptides. Here, we examine these Hsmr TM peptides for stronger (sodium dodecyl sulfate-resistant) and weaker (sodium perfluorooctanoate-resistant) TM-TM interactions, in conjunction with CD and fluorescence energy transfer (FRET) measurements and molecular modeling, to show that helix four has two interactive surfaces that may contribute to the stability of SMR protein oligomers. Peptide Synthesis and Purification—Prediction of Hsmr TM segments was performed using the program TM Finder (18Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar) with all default parameters and with gap length set to zero. Lysine-tagged peptides corresponding to Hsmr residues 2–24 (TM-1), 30–51 (TM-2), 56–79 (TM-3), and 85–105 (TM-4) with the sequences K-HPYAYLAAAIAAEVAGTTALKLS-K, KK-PAPSVVVLVGYVSSFYFLGLVL-KKK, KKK-VGVVYGTWAAVGIVATALVGVVF-KKK, and KKK-VAGVVGLALIVAGVVVLNVAS-KK were synthesized with a Pioneer peptide synthesizer (Applied Biosystems) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry (19Amblard M. Fehrentz J.A. Martinez J. Subra G. Methods Mol. Biol. 2005; 298: 3-24PubMed Google Scholar) on a PAL-PEG-PS 4′-aminomethyl-3′, 5′-dimethoxyphenoxyvaleric acid-poly(ethylene glycol) polystyrene resin (Applied Biosystems) that produced an amidated C terminus upon peptide cleavage. The lysine residues added to each peptide sequence were not anticipated to affect peptide orientation or stoichiometry (20Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar, 21Therien A.G. Deber C.M. J. Biol. Chem. 2002; 277: 6067-6072Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Partridge A.W. Melnyk R.A. Yang D. Bowie J.U. Deber C.M. J. Biol. Chem. 2003; 278: 22056-22060Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Zouzoulas A. Therien A.G. Scanzano R. Deber C.M. Blostein R. J. Biol. Chem. 2003; 278: 40437-40441Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Labeling with 4-dimethylaminoazobenzene -4′-sulfonyl chloride (dabcyl chloride), 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride), or 5- (and 6-) carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE) (Molecular Probes) was accomplished by incubating the resin-bound peptide with excess label under basic conditions overnight. Cleaved peptides were by on a was used to the molecular of the and the was used to determine Peptide on in was performed on of peptide in One peptide was with of peptide in SDS-PAGE to peptide to that of peptides. was performed as M. Biochem. J. PubMed Scopus Google Scholar) on in A peptide of was used in with of peptides was performed as was used on all to peptides and was used in conjunction with to peptides in measurements were performed using CD and in and were in a length on a circular at peptide of in and in a length at peptide of in The for peptides in a was using the is the of residues in the peptide Yang Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). to this the at corresponding to conformations are and a of peptide was with of the corresponding peptide in or and to at overnight. peptide was at by the of the of were obtained in a length by the at an of with a and from to with a at each in the on a were between and using the by the fluorescence at each in the were by the fluorescence in the of peptide was to the were as Engelman D.M. Biochemistry. 1994; PubMed Scopus Google Scholar) using the program a peptide was or and CD and fluorescence measurements were performed in 10 with or with of the between two helices corresponding to Hsmr residues 85–105 (TM-4) were produced as C. Deber C.M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of Hsmr TM a to helix-helix in Hsmr SMR proteins have short loops between TM segments residues in length on helix M. Lebendiker M. Yerushalmi H. Coles M. Groger A. Schwarz C. Schuldiner S. Kessler H. Eur. J. Biochem. 1998; 254: 610-619Crossref PubMed Scopus (89) Google Scholar)) and must be stabilized by intermolecular TM helix-helix and antiparallel helix-helix interactions to be an for SMR oligomers that may assemble with (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar, 9Ubarretxena-Belandia I. Baldwin J.M. Schuldiner S. Tate C.G. EMBO J. 2003; 22: 6175-6181Crossref PubMed Scopus (171) Google Scholar, 11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 12Pornillos O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar). corresponding to each TM segment of Hsmr were with by to in EmrE M. Lebendiker M. Yerushalmi H. Coles M. Groger A. Schwarz C. Schuldiner S. Kessler H. Eur. J. Biochem. 1998; 254: 610-619Crossref PubMed Scopus (89) Google Scholar), the α-helices in EmrE (11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 12Pornillos O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar), and use of the program TM Finder (18Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar). The peptides synthesized residues for lysine residues at their and C the (20Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar) that peptide the in vivo oligomeric stoichiometry and helix-helix of TM segments (20Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar, 21Therien A.G. Deber C.M. J. Biol. Chem. 2002; 277: 6067-6072Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Partridge A.W. Melnyk R.A. Yang D. Bowie J.U. Deber C.M. J. Biol. Chem. 2003; 278: 22056-22060Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Zouzoulas A. Therien A.G. Scanzano R. Deber C.M. Blostein R. J. Biol. Chem. 2003; 278: 40437-40441Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). of Hsmr TM in of the four Hsmr peptides in the and of or are in the of which a minimum each Hsmr TM peptide CD with a in The and peptides α-helical CD with at and in the of or with the of SMR The of a minimum at in and and of its in the self-assembly of Hsmr was not and of Hsmr TM in of the peptides was by SDS-PAGE is and is in the that the in SMR proteins M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google Scholar), although for (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar), may not stabilize the SDS-resistant Hsmr that the molecular of the vary and and their on the are thus not although and oligomeric under the peptide is and has been as an intermolecular in EmrE M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google Scholar). of The peptide to discrete dimers and to the of in Hsmr in the of this of and were for their to SDS-resistant or peptides were and in with peptides with the of the peptide with its is of (20Melnyk R.A. Partridge A.W. Deber C.M. Biochemistry. 2001; 40: 11106-11113Crossref PubMed Scopus (84) Google Scholar). peptide a to its and of and peptides was not for of the peptides and We although intermolecular have been in EmrE O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar, M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google Scholar), of not be SDS-resistant dimer in and of Hsmr TM in of the and peptides to and in was also is a that helix-helix interactions denatured by A.G. Deber C.M. J. Biol. Chem. 2002; 277: 6067-6072Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and can the of membrane proteins M. Biochem. J. PubMed Scopus Google Scholar). We to the SDS-resistant dimer and also permit the of lower affinity helix-helix interactions among the Hsmr peptides. of and in was with SDS-PAGE The as a strong to resist in A.G. Deber C.M. J. Biol. Chem. 2002; 277: 6067-6072Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, M. Biochem. J. PubMed Scopus Google Scholar), we that via at least two sets of intermolecular interactions, that is but in PFO, and that is to by both and and for on were performed on both and and TM peptides C and labeling not affect of and with peptide and of the and peptides was also and and and was not for all of and peptides and results are with two the of that not or or interactions are of lower affinity by stoichiometry of oligomerization was in in or these of the peptides of peptide results in energy transfer and a of the group fluorescence by the group Engelman D.M. Biochemistry. 1994; PubMed Scopus Google Scholar). fluorescence is for peptides that are a between fluorescence and peptide is for and a is of higher oligomers Engelman D.M. Biochemistry. 1994; PubMed Scopus Google Scholar). in the fluorescence between and from the peptide at was upon with an of in or that is and as a for oligomerization in the The in peptide fluorescence upon with peptide in is of dimer of and to a stoichiometry of with an of the in fluorescence in to a stoichiometry of with an of the stoichiometry for by SMR proteins in vivo Schuldiner S. EMBO J. 2000; 19: PubMed Scopus Google Scholar) and as an between dimers and Y. Steiner-Mordoch S. Danieli T. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1519-1524Crossref PubMed Scopus (125) Google Scholar). of by the of the peptide to oligomers with must have at least two helix surfaces of intermolecular interactions as oligomerization a dimer a on each helix two helix surfaces in a or symmetric with their the stoichiometry of the resulting multimer is to which be on as of the peptide to oligomers of is not in sets of conditions in which stoichiometry can be to a discrete symmetric interactions are not and each in asymmetric or a of symmetric and asymmetric is Our for the of was by evidence for symmetric in oligomeric EmrE M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google Scholar) on a that we have the The of this in self-assembly was with mutant peptides in which two and as as an were in with their corresponding residues from the EmrE dimer is denatured in (13Ninio S. Elbaz Y. Schuldiner S. FEBS Lett. 2004; 562: 193-196Crossref PubMed Scopus (48) Google Scholar), these were to disrupt The resulting and CD to not and all but the affinity of dimers on SDS-PAGE by the of a by measurements show that the and to of of these also or fluorescence with in of monomers M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google Scholar) to a peptide an to on SDS-PAGE and in residues are of SDS-resistant helix-helix association H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), we that the dimer was stabilized via strong der interactions Deber C.M. Biochemistry. 2004; PubMed Scopus Google of Hsmr in SDS-PAGE of and mutant peptides. and on the and are to the Although the mutant is at a to the other by in not and as a less fluorescence of and mutant peptides with corresponding peptides in with the of association of the peptide with the peptide was The of was to be lower for this of peptides as each has a at of the two interactive helix surfaces on each the standard of at least and the best to the between the of in and the of dimer on SDS-PAGE of on by of peptide as dimers or monomers on SDS-PAGE with were obtained by the at the monomer or dimer by the of the at both by of peptide as dimers or monomers on SDS-PAGE with were obtained by the at the monomer or dimer by the of the at both in a The Hsmr sequence was thus for that can the of stabilized by der Waals Deber C.M. Biochemistry. 2004; PubMed Scopus Google Scholar). of these, a Engelman D.M. 2001; PubMed Scopus (49) Google Scholar) and and a Y. Engelman D.M. M. Scholar) and were on a distinct from the that we have the are by the highly of to was by the oligomeric state of peptides and or The was on the EmrE sequence and were with was to of as small residues such as are compatible with Deber C.M. Biochemistry. 2004; PubMed Scopus Google Scholar), and of the to was anticipated to be of these CD to in not and a or and to A and We that residues were also in the of the SDS-resistant It is to that that the sequence of SMR family members and on the and on the the affinity of, but not The of dimer on SDS-PAGE also with the of the for the and mutant peptides The of Hsmr or helix the and the thus to be in these faces as in mutant peptides with or on their be to with as each a A mutant was with an mutant these fluorescence of the peptide as the peptide is the in the The in fluorescence from the peptide with the in thus provides evidence that between and is We also the of each mutant to with in peptide is added to an of of the fluorescence of the peptide is with fluorescence was upon of interactions with the peptides not in each mutant was with the in these fluorescence with was that a helix of SDS-resistant with is in peptides with on the or the The of Hsmr via and asymmetric between helices are the interactions all the intermolecular surfaces in the be and from to be on and are not may be oligomerization as in via an (and asymmetric between the and along with an but the in this interaction, that are highly and thus on SDS-PAGE and be to be on The and mutant are not monomers We that is the in the and that their in PFO, in of the symmetric interactions between EmrE residues in M. Steiner-Mordoch S. Schuldiner S. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus (77) Google The of is with this both dimers and on that can be by the assembly of asymmetric dimers on that symmetric pairing at the to in the but not the asymmetric pairing in or the symmetric in PFO, resulting in a The and not in PFO, with of but of interactions, at lower affinity WT, in We that the on the sequence of EmrE, not all and dimer in are of SMR family sequence and are compatible with The and may oligomerization in and in peptide the topology of interactions, molecular was used to determine whether the asymmetric and symmetric from were in antiparallel helix with energy conformations for dimers asymmetric or symmetric were not Although we the of oligomerization via or antiparallel that the TM helix of the H. salinarum SMR protein has two distinct interactive surfaces that in a or asymmetric to assemble a affinity dimer and a discrete via a lower affinity symmetric of self-assembly to be by membrane proteins to oligomerization. and C. M. in the affinity that we in the Hsmr to the stability of SMR family an model of assembly of the in of the SMR monomer M. Schuldiner S. Kessler H. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and a in the SMR monomer that (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar). this in the structural inequivalence of the two subunits in the SMR dimer I. Baldwin J.M. Schuldiner S. Tate C.G. EMBO J. 2003; 22: 6175-6181Crossref PubMed Scopus (171) Google Scholar, 10Tate C.G. Ubarretxena-Belandia I. Baldwin J.M. J. Mol. Biol. 2003; 332: 229-242Crossref PubMed Scopus (71) Google Scholar, 11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 12Pornillos O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar) is by an defined as a at the in a from to pairing with the folds are in model for assembly of the asymmetric SMR dimer via and also for, but not symmetric a tetramer. Although the of model of to SMR assembly to be in the structural studies that has surfaces O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar) and that the helix-helix interactions in SMR family proteins can vary (11Ma C. Chang G. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2852-2857Crossref PubMed Scopus (77) Google Scholar, 12Pornillos O. Chen Y.J. Chen A.P. Chang G. Science. 2005; 310: 1950-1953Crossref PubMed Scopus (71) Google Scholar). of SMR assembly must also the in EmrE (5Ubarretxena-Belandia I. Tate C.G. FEBS Lett. 2004; 564: 234-238Crossref PubMed Scopus (35) Google Scholar), of to dimer and the of on the The and of SMRs is a process that must helix-helix interactions that can vary from monomer to a that may to the required for a protein and thus have for the SMR mechanism. oligomerization of SMR proteins is required for their drug efflux of as a of SMR dimers provides a for design of therapeutics that target this multidrug resistance mechanism. We S. Schuldiner for and on an of this

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