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Originally published In Press as doi:10.1074/jbc.M600434200 on March 30, 2006

J. Biol. Chem., Vol. 281, Issue 22, 15546-15553, June 2, 2006
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Evidence for Assembly of Small Multidrug Resistance Proteins by a "Two-faced" Transmembrane Helix*

Arianna Rath{ddagger}§1, Roman A. Melnyk{ddagger}§2, and Charles M. Deber{ddagger}§3

From the {ddagger}Division of Structural Biology & Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the §Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Received for publication, January 17, 2006 , and in revised form, March 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The advances made in controlling and treating infectious diseases using antibiotics are threatened by the increase of multidrug resistance in pathogenic organisms (1). 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 (2). Among these, the small multidrug resistance (SMR)4 proteins are highly prevalent (>60 homologs in both Gram-positive and Gram-negative bacteria (3)) proton/drug antiporters of 100–110 amino acids (4). SMRs catalyze the efflux of large (up to 10 Å cross-section), structurally diverse hydrophobic cations such as acriflavine, ethidium, methyl viologen, tetracycline, and tetraphenylphosphonium (5). The monomer of the best characterized family member, the Escherichia coli SMR protein EmrE, is a tightly packed bundle of four antiparallel transmembrane (TM) {alpha}-helices with short loops (6, 7).

The minimum structural unit of EmrE is a dimer that is unusual in that its component monomers have distinct conformations (8–11). The structural inequivalence of these subunits may relate to an antiparallel insertion of individual EmrE polypeptides (5, 9, 11, 12), although evidence for antiparallel topology was not obtained in cysteine accessibility and labeling studies (13). 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 (11, 14), drug efflux activity requires the assembly of two SMR molecules (15). 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 {alpha}-helical TM segment (16). The intermolecular helix-helix interactions that assemble SMR oligomers vary in strength among family members (17) 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 (17). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide Synthesis and Purification—Prediction of Hsmr TM segments was performed using the program TM Finder (18) 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 (19) 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 (20–23). 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 purified by reverse phase-high pressure liquid chromatography on a C4 preparative column (Phenomenex). Mass spectrometry was used to confirm the molecular weight of the purified peptides, and the Micro BCA assay (Pierce) was used to determine concentration.

Peptide Electrophoresis—SDS-PAGE on precast NuPAGE gels containing 12% acrylamide in MES buffer (Invitrogen) was performed on 2 µg of peptide samples in homooligomerization experiments. One µgof unlabeled peptide was mixed with 1 µg of TAMRA-labeled peptide samples before loading gels in SDS-PAGE heterooligomerization experiments to keep total peptide concentration identical to that of unlabeled peptides. Sodium perfluorooctanoate (PFO)-PAGE was performed as described (24) on precast Tris-Glycine gels containing 18% acrylamide in Tris-Glycine buffer (Invitrogen). A total peptide concentration of 2 µg was used in PFO-PAGE experiments with unlabeled peptides; heterooligomerization of labeled peptides was performed as described above. Coomassie Blue staining was used on all gels to visualize peptides and was used in conjunction with UV transillumination to detect TAMRA-labeled peptides in heterooligomerization experiments. Densitometry measurements were performed using NIH Image.

CD and Fluorescence Measurements—CD spectra in buffer and SDS were recorded in a 0.1-cm path length cuvette on a Jasco J-720 circular dichroism spectropolarimeter at peptide concentrations of 20–50 µM in SDS and in a 0.01-cm path length cuvette at peptide concentrations of 50–100 µM in PFO. The mean residue ellipticity expected for peptides in a 100% helical conformation was calculated using the formula

Formula(Eq. 1)
where n is the number of residues in the peptide (25, 26). According to this relation, the mean residue ellipticities (deg cm2 dmol–1 res–1) at 222 nm corresponding to 100% helical conformations are –32,640 (TM-1); –33,185 (TM-2); –33,867 (TM-3); and –32,923 (TM-4).

For FRET quenching experiments, a 1 µM solution of dansyl-labeled peptide (donor fluor) was mixed with 0–4µM of the corresponding dabcyl-labeled peptide (acceptor) in SDS or PFO micelles and allowed to equilibrate at room temperature overnight. Total peptide concentration was kept constant at 5 µM by the addition of the appropriate concentration of unlabeled peptide. Emission spectra were obtained in a 1-cm path length cuvette by exciting the dansyl fluor at an excitation wavelength of 341 nm with a 2 nm bandpass and monitoring emission from 450 to 650 nm with a 4 nm bandpass at each point in the titration on a Photon Technology International C-60 spectrofluorimeter. Spectra were integrated between 450 and 650 nm using the FELIX software provided by the manufacturer. Integrated total fluorescence intensities at each point in the titration (F) were normalized by the initial integrated total fluorescence intensity in the presence of 0 µM acceptor peptide (Fo). Mole fraction acceptor (Pa) was calculated according to the relation

Formula(Eq. 2)
Data were fit as described (27) using the program Kaleidagraph. In FRET competition experiments, a 1 µM dansyl-labeled peptide solution was mixed with: 1 µM unlabeled peptide; 1 µM dabcyl-labeled peptide; or 1 µM dabcyl and 2 µM unlabeled peptide. CD and fluorescence measurements were performed in buffer (10 mM Tris, pH 7.2, 10 mM NaCl), buffer with 20 mM SDS, or buffer with 50 mM PFO.


Figure 1
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  		FIGURE 1.
CD spectra of WT Hsmr TM peptides. A, CD spectra in aqueous buffer containing 10 mM Tris, pH 7.2, and 10 mM NaCl and in this buffer with 20 mM SDS (B) or 50 mMPFO (C). All labeled peptides and Hsmr TM-4 mutants similarly displayed {alpha}-helical spectra under these conditions. Concentrations for TM-1, TM-3, and TM-4 were adjusted based on gel values for oligomer stoichiometry in SDS or PFO micelles.

 
Molecular Modeling—Energy-minimized models of the interaction between two helices corresponding to Hsmr residues 85–105 (TM-4) were produced as described (28).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Design of Hsmr TM Peptides—We selected a peptide-based approach to evaluate helix-helix contacts in Hsmr because SMR proteins have short connecting loops between TM segments (5–9 residues in length based on NMR helix boundary assignments (7)) and must therefore be predominantly stabilized by intermolecular TM helix-helix contacts. This technique permits parallel and antiparallel helix-helix interactions to be detected, an ideal methodology for SMR oligomers that may assemble with inverted topologies (5, 9, 11, 12). Peptides corresponding to each WT TM segment of Hsmr were initially synthesized, with boundaries selected by referring to NMR {alpha}-helix assignments in EmrE (7), the {alpha}-helices observed in EmrE x-ray structures (11, 12), and use of the TM-segment predicting program TM Finder (18). The peptides synthesized (TM-1, residues 2–24; TM-2, 36–55; TM-3, 56–79; TM-4, 85–105; see "Experimental Procedures" for sequences) incorporate multiple lysine residues at their N and C termini following the "Lys tag" technique (20) that increases peptide solubility while preserving the in vivo oligomeric stoichiometry and native helix-helix interfaces of TM segments (20–23).


Figure 2
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  		FIGURE 2.
Homo- and heterooligomerization of WT Hsmr peptides on SDS-PAGE. A, SDS-PAGE on gels containing 12% acrylamide of 2 µg of WT TM-1, TM-3, and TM-4 peptides, visualized by staining with Coomassie Blue. The molecular mass of each peptide is as follows: TM-1, 2.56 kDa; TM-3, 3.12 kDa; TM-4, 2.56 kDa. Oligomeric states are indicated to the right of the figure. See "Results" for further discussion. B and C, Coomassie Blue stain (B) and UV transillumination (C) of mix and match SDS-PAGE on gels containing 12% acrylamide of TAMRA-labeled and unlabeled WT peptides. Unlabeled peptide is indicated under each lane, and TAMRA (TAM)-labeled peptide is indicated by labels under the black bars. The apparent increase in molecular weight observed for the TAM-TM-4 peptide in panel C, lane 9, results from the higher peptide concentration (33) (2 µg versus 1 µg in lanes 7–8). The apparent downshift in the molecular mass of TM-4-TAM visualized by Coomassie Blue staining in the presence of TM-1 or TM-3 in panel B, lanes 7–8, similarly results from the 1 µg concentration of TM-4 combined with the slightly altered mobility of TM-4-TAM when compared with TM-4 at equivalent concentrations (compare panel A, lane 3 to panels B and C, lane 9).

 


Figure 3
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  		FIGURE 3.
Homo- and heterooligomerization of WT Hsmr TM peptides on PFO-PAGE. A, PFO-PAGE of unlabeled WT Hsmr TM peptides. B, mix and match PFO-PAGE of unlabeled WT Hsmr TM peptides. A solid bar under the gel lanes denotes peptide mixtures; the peptide indicated under the bar was mixed with the unlabeled peptide indicated under each lane. C and D, Coomassie Blue stain (C) and UV transillumination (D) of mix and match PFO-PAGE of TAMRA-labeled and unlabeled WT peptides. Unlabeled peptide is indicated under each lane, and TAMRA (TAM)-labeled peptide is indicated by labels under the black bars. Oligomeric states are indicated to the right of the gels.

 
Folding of Hsmr TM Peptides in Membrane-mimetic Environments—CD spectra of the four WT Hsmr peptides in the presence and absence of SDS or PFO micelles are shown in Fig. 1. With the exception of TM-2, which had a broad minimum centered near 210 nm, each Hsmr TM peptide displayed CD spectra consistent with a random-coil conformation in aqueous buffer (Fig. 1A). The TM-1, TM-3, and TM-4 peptides had {alpha}-helical CD spectra with minima at 208 and 222 nm in the presence of SDS (Fig. 1B) or PFO (Fig. 1C), secondary structure consistent with the conformation of native SMR proteins. The spectrum of TM-2, however, had a broad minimum centered at ~217 nm in SDS and PFO micelles (Fig. 1, B and C), characteristic of beta-strand conformation; its role in the self-assembly of Hsmr was therefore not investigated further.

Homo- and Heterooligomerization of Hsmr TM Peptides in SDS—Oligomerization of the TM-1, TM-3, and/or TM-4 peptides was evaluated by SDS-PAGE (Fig. 2). TM-1 is monomeric and TM-4 is dimeric in the 12% NuPAGE system (Fig. 2A), suggesting that the TM-1/TM-1 contacts described in SMR proteins (29), although important for function (5), may not stabilize the SDS-resistant Hsmr oligomer. Note that the molecular masses of the three constructs vary (TM-1 and TM-4, 2.56 kDa; TM-3, 3.12 kDa), and their migration rates on the gel are thus not comparable. Further, although TM-1 and TM-3 report oligomeric states >1.0 under some conditions, the TM-4 peptide is consistently dimeric (Fig. 2A) (or tetrameric (vide infra)) throughout our experiments and has been identified as an intermolecular contact site in EmrE (29). This intriguing ability of The TM-4 peptide to form discrete dimers and tetramers led us to explore the role of TM-4 in Hsmr stabilization in the remainder of this study.

All combinations of TM-1, TM-3, and TM-4 were tested for their ability to form SDS-resistant heterooligomers. Unlabeled TM-1, TM-3, or TM-4 peptides were "mixed and matched" in equimolar ratios with peptides labeled with the UV-fluorescent dye TAMRA; here, co-migration of the labeled peptide with its unlabeled counterpart is indicative of heterooligomerization (20). Each TAMRA-labeled peptide had a migration pattern similar to its unlabeled counterpart, and co-migration of unlabeled and TAMRA-labeled peptides was not observed for any combination of the TM-1, TM-3, and/or TM-4 peptides (compare Fig. 2B and 2C). We therefore concluded that, although intermolecular TM-1/TM-4 contacts have been described in EmrE (12, 29), heterooligomeric pairs of TM-1, TM-3, and/or TM-4 might not be mediating SDS-resistant dimer formation in Hsmr.

Homo- and Heterooligomerization of Hsmr TM Peptides in PFO—The ability of the TM-1, TM-3, and TM-4 peptides to homo- and heterooligomerize in PFO micelles was also tested (Fig. 3). PFO is a "mild" detergent that preserves helix-helix interactions denatured by SDS (21), and PFO-PAGE can preserve the native quaternary structures of membrane proteins (24). We therefore expected PFO to preserve the SDS-resistant TM-4 dimer and also permit the formation of any lower affinity helix-helix interactions among the Hsmr peptides. Oligomerization of TM-1 and TM-3 in PFO-PAGE was unchanged when compared with SDS-PAGE (Fig. 3A). The TM-4 peptide, however, migrated as a tetramer (Fig. 3A). Since any interaction strong enough to resist SDS denaturation should persist in PFO (21, 24), we hypothesized that TM-4 self-assembles via at least two sets of intermolecular interactions, one that is SDS-sensitive, but permitted in PFO, and another that is resistant to denaturation by both SDS and PFO.

"Mix and match" experiments for heterooligomerization on PFO-PAGE were performed on both unlabeled (Fig. 3B) and TAMRA-labeled and unlabeled TM peptides (Fig. 3, C and D). TAMRA labeling did not affect gel migration of TM-1, TM-3, and TM-4 when compared with unlabeled peptide (compare Fig. 3A and 3D). Migration of the unlabeled TM-1, TM-3, and TM-4 peptides was also unchanged when mixed and matched (compare Fig. 3A and 3B), and co-migration was not observed for all possible combinations of TAMRA-labeled and unlabeled peptides (compare Fig. 3C and 3D). These results are consistent with two possibilities: either the surface(s) of TM-4 that mediate homooligomerization do not bind TM-1 and/or TM-3 or any potential TM-1/TM-4 or TM-3/TM-4 interactions are of lower affinity than TM-4/TM-4 self-associations.

TM-4 Stoichiometry Determined by Fluorescence Energy Transfer—The stoichiometry of TM-4 oligomerization was tested in FRET experiments in SDS or PFO micelles (Fig. 4). In these assays, co-localization of the peptides within 30 Å, indicative of peptide oligomerization, results in energy transfer and a stoichiometry-dependent quenching of the dansyl group fluorescence by the dabcyl acceptor group (27). Little fluorescence quenching is expected for peptides that are monomeric, a linear relationship between dansyl fluorescence yield and acceptor peptide concentration is expected for dimers, and a non-linear relationship is indicative of higher order oligomers (27).

Within experimental error, no change in the total fluorescence yield between 450 and 650 nm from the dansyl-TM-1 peptide after excitation at 341 nm was observed upon titration with an increasing proportion of dabcyl-TM-1 in SDS (Fig. 4A) or PFO (Fig. 4B), confirming that TM-1 is monomeric and acting as a negative control for oligomerization in the FRET assay. The linear decrease observed in dansyl-TM-4 peptide fluorescence upon titration with acceptor peptide in SDS is diagnostic of dimer formation of and fits to a stoichiometry of 2.3 ± 0.1 units/oligomer with an R-value of 0.996 (Fig. 4A), whereas the non-linear decrease in dansyl-TM-4 fluorescence yield observed in PFO micelles fits to a stoichiometry of 3.2 ± 0.2 units/oligomer with an R-value of 0.998 (Fig. 4B). These PFO data mirror the 3:1 protein:substrate stoichiometry described for ligand binding by SMR proteins in vivo (30) and interpreted as representing an equilibrium between dimers and tetramers (14).


Figure 4
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  		FIGURE 4.
Oligomer stoichiometry of Hsmr TM-4 in FRET experiments. A and B, fluorescence quenching of WT dansyl-TM-1 (circles) or WT dansyl-TM-4 (squares) donor peptides with the corresponding dabcyl-labeled acceptor peptide in 10 mM Tris, pH 7.2, 10 mM NaCl with 20 mM SDS (A) or 50 mM PFO (B). Dansyl-labeled donor peptides were excited at 341 nm, and emission was measured from 450 to 650 nm. Plots were generated and data were fit as described under "Experimental Procedures," and the lines shown represent the best fit to each data set. The stoichiometry of each TM-4 oligomer (n) and the R-values of fits to the TM-4 titration data are indicated on each graph. Error bars correspond to the standard deviation of at least three experiments.

 
Formation of Tetramers by TM-4 Requires the Interaction of Two Helix Faces—For the TM-4 peptide to form oligomers with greater than dimeric stoichiometry, it must have at least two helix surfaces capable of intermolecular interactions as oligomerization cannot proliferate beyond a dimer if a single interaction-competent face exists on each TM-4 helix (Fig. 5A). However, if two helix surfaces interact in a pair-wise or symmetric manner with their counterparts, the stoichiometry of the resulting multimer is difficult to control (Fig. 5B). Such unchecked oligomerization, which would be visible on gels as "laddering" of the TM-4 peptide to oligomers of undefined size, is not observed in our experiments. Two sets of conditions exist in which TM-4 stoichiometry can be restricted to a discrete tetramer: (i) if symmetric interactions are not possible, and each interaction-competent face participates in asymmetric contacts (Fig. 5C) or (ii) if a limited combination of symmetric and asymmetric contacts is permitted (Fig. 5D).


Figure 5
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  		FIGURE 5.
Restriction of homooligomerization stoichiometry by numbers of interacting faces of membrane protein TM helices. Circles represent an axial view of a single TM helix perpendicular to the bilayer plane. Helix faces capable of intermolecular interactions are indicated in green and gold. A, each helix has a single interaction-competent face (gold) capable of pairing with its counterpart in another helix. Once the two faces are paired, the intermolecular interaction potential of each helix is satisfied, and stoichiometry is limited to dimer. B, each helix has two distinct interaction-competent faces (gold and green) that can interact with their counterparts in another helix in a pairwise or symmetric manner, such that gold/gold and green/green pairs form. Because there is always an unpaired gold and/or green face at the oligomer edges, stoichiometry is undefined, and infinite oligomerization can occur. C, the two helix faces interact in an asymmetric manner, such that only gold/green pairings are permitted. Stoichiometry is restricted because the oligomer eventually cyclizes, and all four intermolecular interaction interfaces (gold/green) are equivalent. D, a single helix face (gold) is capable of symmetric (gold/gold) and asymmetric (gold/green) intermolecular interactions. Stoichiometry is restricted because green/green pairings are not possible, and exposure of interactive surfaces at the oligomer edges is minimized. The resulting oligomer contains two non-equivalent intermolecular interaction interfaces (gold/gold and gold/green).

 


Figure 6
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  		FIGURE 6.
TM-4 sequences of SMR proteins. A helical wheel projection of the Hsmr TM-4 sequence with the Ala-Val face and the Gly face is shown with an alignment of the Hsmr, EmrE, and SMR consensus (36) TM-4 sequences. Conserved residues are boxed, and substituted residues on the Ala-Val face and Gly face are shown in gold and green, respectively. The EmrE sequence given is that of the "Cys-less" mutant shown to have WT activity (29), used here as a basis for residue substitutions to eliminate disulfide bond formation.

 
Our search for the contact site(s) of TM-4 was guided by evidence for symmetric TM-4/TM-4 contacts observed in oligomeric EmrE (29) on a surface that we have termed the "Ala-Val face" (Fig. 6). The role of this surface in TM-4 self-assembly was assessed with mutant peptides in which two Ala-Val face residues, Ala-92 and Val-95, as well as an adjacent residue, Val-85, were substituted singly and/or in combination with their corresponding residues from EmrE. Because the EmrE dimer is denatured in SDS (13), these replacements were expected to disrupt TM-4 dimerization. The resulting mutants, A92M, V95S, A92M/V95S, and V85L, had CD spectra essentially identical to WT TM-4 (data not shown), and all but V85L reduced the affinity of TM-4 dimers on SDS-PAGE gels, evidenced by the appearance of a monomeric species (Fig. 7A). Relative band intensities determined by densitometry measurements show that the A92M, V95S, and A92M/V95S mutants reduce dimerization to ~50% of WT levels (Table 1). Each of these mutants also exhibited decreased or eliminated fluorescence quenching when compared with WT in FRET experiments (Fig. 7B). Not every Ala-Val face residue capable of bridging monomers (29) contributes to TM-4/TM-4 affinity, however; a peptide containing an Asn-102 to Ala replacement (Fig. 6) remained dimeric on SDS-PAGE (Fig. 7A) and in FRET experiments (Fig. 7B). As Asn residues are typical mediators of SDS-resistant helix-helix association (31, 32), we hypothesized that the TM-4 dimer was stabilized via strong van der Waals, rather than electrostatic, interactions (33).


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  		TABLE 1
Dimerization of TM-4 mutants on SDS-PAGE

 
The Hsmr TM-4 sequence was thus searched for motifs that can facilitate the "knobs-into-holes" contacts characteristic of interfaces stabilized by van der Waals forces (33). Two of these, a leucine zipper-like motif (34) (Leu-93 and Leu-101) and a GG7 motif (35) (Gly-90 and Gly-97), were found on a TM-4 surface distinct from the Ala-Val face that we have designated the "Gly face," where they are flanked by the highly conserved residue Ile-94 (Fig. 6). Contribution of Gly face contacts to TM-4 dimerization was evaluated by determining the oligomeric state of peptides containing disrupted motifs (L101I and G90V) or conserved residue replacements (I94A). The L101I substitution was selected based on the EmrE TM-4 sequence (Fig. 6), whereas Gly-90 and Ile-94 were replaced with non-conservative residues. G90V was selected to ensure perturbation of any Gly face-mediated contacts as small residues such as Ala are compatible with knobs-into-holes packing (33), and truncation of the Ile-94 side chain to Ala was similarly anticipated to be disruptive. Each of these mutants had CD spectra nearly identical to WT in SDS micelles (data not shown) and had a reduced (L101I) or completely abrogated (G90V and I94A) ability to dimerize (Fig. 7, A and B, Table 1). We therefore concluded that Gly face residues were also involved in the formation of the SDS-resistant TM-4/TM-4 dimers. It is interesting to note that those substitutions that keep the TM-4 sequence representative of SMR family members (A92M and V92S on the Ala-Val face and L101I on the Gly face) reduce the affinity of, but do not completely abolish, dimerization. The percentage of dimer observed on SDS-PAGE also correlates with the slope of the quenching curve for the TM-4 WT and mutant peptides (r = 0.850, Fig. 7C).


Figure 7
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  		FIGURE 7.
Homooligomerization of Hsmr TM-4 mutants in SDS. A, SDS-PAGE of WT and mutant TM-4 peptides. Gold and green asterisks indicate residue substitutions on the Ala-Val face and Gly face, respectively. Oligomeric states are indicated to the right. Although the V95S mutant is loaded at a concentration equivalent to the other peptides, it consistently displays reduced staining by Coomassie Blue in SDS gels (data not shown) and therefore appears as a less intense band. B, fluorescence quenching of dansyl-labeled WT and mutant TM-4 peptides with corresponding dabcyl-labeled peptides in SDS micelles with the exception of I94A-A92M/V95S, where association of the dansyl-I94A peptide with the dabcyl-A92M/V95S peptide was monitored. The percentage of quenching was expected to be lower than WT for this pair of peptides as each has a lesion at one of the two interactive helix surfaces on each helix. Error bars represent the standard deviation of at least three independent experiments, and lines represent the best fit to the data. C, Correlation between the slope of quenching curves in panel B and the percentage of dimer observed on SDS-PAGE gels.

 
The TM-4 of Hsmr Forms Asymmetric or "Two-faced" Dimers—Two TM-4 helix surfaces, the Ala-Val face and the Gly face, thus appear to be involved in dimerization. If these faces contact asymmetrically, as in Fig. 5C, then mutant peptides with one or more lesions on their Ala-Val face should be able to interact with Gly face mutants as each retains a single interaction-competent surface. A Gly face mutant donor peptide, dansyl-I94A, was accordingly titrated with an Ala-Val face mutant acceptor peptide, dabcyl-A92M/V95S. Under these conditions, fluorescence quenching of the dansyl-I94A peptide indicates heterooligomer formation as the dabcyl-A92M/V95S peptide is the sole acceptor present in the titration. The linear decrease in fluorescence from the dansyl-I94A donor peptide when mixed with the dabcyl-A92M/V95S acceptor in FRET titration experiments (Fig. 7B) thus provides evidence that dimerization between Ala-Val face and Gly face mutants is possible.


Figure 8
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  		FIGURE 8.
Disruption of WT dimers by "single face" mutants of TM-4. A, fluorescence emission spectra of WT dansyl-labeled TM-4 peptide excited at 341 nm in the presence of unlabeled WT TM-4 (black); WT dabcyl-TM-4 (red); or in the presence of WT dabcyl-TM-4 and unlabeled WT TM-4 (blue) peptides. B, disruption of the WT TM-4 dimer by monomeric single face mutant peptides. The total area under the emission spectrum from 450 to 650 nm of each peptide mixture in SDS micelles is shown normalized to the fluorescence of an unquenched solution. Error bars represent the standard deviation of three experiments. WT dansyl-TM-4 was mixed with unlabeled WT TM-4 (black); dabcyl-TM-4 (red); or dabcyl-TM-4 and the indicated WT (blue), Ala-Val face mutant (gold), Gly face mutant (green) or WT TM-1 (gray) peptide.

 


Figure 9
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  		FIGURE 9.
PFO-PAGE of WT and mutant TM-4 peptides. TM-1 was run on the gel as a monomeric control. Bands migrating at a larger molecular weight than the WT tetramer are present in the lanes corresponding to the V95S and N102A mutant peptides. Gold and green asterisks indicate residue substitutions on the Ala-Val face and Gly face, respectively. Oligomers are indicated to the right.

 


Figure 10
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  		FIGURE 10.
Interface switching model of functional SMR oligomer assembly. The SMR monomer is represented as a four-helix bundle with the TM-4 Ala-Val face (gold) and Gly face (green) shown. A, initial contact between SMR monomers. The Gly face is buried, and the Ala-Val faces may form symmetric contacts. B, the Gly face is exposed by a structural change in one monomer, shown here as a rotation for illustration purposes, and contacts the Ala-Val face in the other monomer to form an asymmetric dimer. The remaining Ala-Val face in this dimer (C) is then available to assemble a tetramer (D) via symmetric pairing with the Ala-Val face in another asymmetric dimer (related by a 180° rotation to the dimer in panel B) that cannot proliferate to higher oligomeric states.

 
We also tested the ability of each TM-4 mutant to heterooligomerize with WT TM-4 in FRET competition experiments (Fig. 8). When unlabeled WT TM-4 peptide is added to an equimolar mixture of dansyl-TM-4/dabcyl-TM-4, ~50% of the fluorescence of the dansyl-labeled peptide is recovered when compared with unquenched dansyl-TM-4 (Fig. 8A). Conversely, no fluorescence recovery within experimental error was detected upon competition of WT dansyl-TM-4/dabcyl-TM-4 interactions with TM-1 (Fig. 8B); the peptides do not heterooligomerize in SDS. However, when each TM-4 mutant was mixed with the WT dansyl-TM-4/dabcyl-TM-4 homodimer in these experiments, fluorescence recovery comparable with WT TM-4 was observed (Fig. 8B), implying that a helix face capable of SDS-resistant interaction with WT is preserved in peptides with lesions on either the Ala-Val face or the Gly face.

The TM-4 of Hsmr Tetramerizes via Asymmetric and Symmetric Interactions—If asymmetric Ala-Val face/Gly face contacts between TM-4 helices are the sole interactions mediating assembly, then all the intermolecular interaction surfaces in the tetramer should be identical (Fig. 5C), and "all-or-none" oligomerization, denaturation from tetramer to monomer, should be observed. However, on PFO-PAGE, Ala-Val face and Gly face mutants are not monomeric (Fig. 9). This result may be rationalized if TM-4 oligomerization proceeds as in Fig. 5D, via an SDS- (and PFO-) resistant asymmetric interaction between the Ala-Val face and Gly face along with an additional PFO-resistant, but SDS-sensitive, interaction. If the Gly face participates in this second interaction, then TM-4 mutants that are highly disruptive and thus monomeric on SDS-PAGE (G90V and I94A) might be expected to be monomeric on PFO-PAGE. The G90V and I94A mutant peptides, however, are not monomers (Fig. 9). We therefore suspect that it is the intact Ala-Val face in the G90V and I94A mutants that mediates their homodimerization in PFO, particularly in light of the symmetric interactions previously detected between EmrE Ala-Val face residues in mild detergent (n-dodecyl-beta-maltoside (29)). The behavior of our mutants is consistent with this model. For example, Leu-101 forms both dimers and tetramers on PFO-PAGE (Fig. 9) that can be explained by the assembly of residual SDS-and PFO-resistant asymmetric Ala-Val face/Gly face dimers (observed on SDS-PAGE, Fig. 7A) that then utilize symmetric pairing at the intact Ala-Val face to mediate tetramerization in PFO. Similarly, the A92M replacement disrupts, but does not completely eliminate, the asymmetric Ala-Val face/Gly face pairing in SDS (Fig. 7A) or the symmetric Ala-Val face/Ala-Val face contacts in PFO, resulting in a dimer-tetramer equilibrium (Fig. 9). The V95S and A92M/V95S mutants do not tetramerize in PFO, consistent with disruption of Ala-Val face/Ala-Val face contacts but preservation of Ala-Val face/Gly face interactions, albeit at lower affinity than WT, in SDS (Fig. 7A). We suspect that the Ala-Val face substitutions, selected based on the TM-4 sequence of EmrE, do not abolish all tetramer and dimer formation in PFO because they are representative of native SMR family sequence and are therefore compatible with self-assembly. The V95S and N102A substitutions, however, may promote non-native oligomerization in PFO (Fig. 9).

Asymmetric and Symmetric Interfaces Are Detected in TM-4/TM-4 Modeling—Because our peptide data cannot resolve the topology of TM-4/TM-4 interactions, molecular modeling was used to determine whether the asymmetric Ala-Val face/Gly face contacts and symmetric Ala-Val face pairs inferred from our experiments were selectively preferred in parallel versus antiparallel helix orientations. Simulations with either topology, however, returned nearly identical low energy conformations for TM-4 dimers where either asymmetric Ala-Val face/Gly face contacts or symmetric Ala-Val face contacts were present (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although we cannot eliminate the possibility of oligomerization via parallel or antiparallel topology, our experiments suggest that the fourth TM helix of the H. salinarum SMR protein has two distinct interactive surfaces that associate in a two-faced or asymmetric manner to assemble a high affinity dimer and build a discrete tetramer via a lower affinity symmetric interaction. This two-faced mode of self-assembly appears to be more generally exploited by membrane proteins to control oligomerization.5 If the two-faced high affinity interaction that we detect in the Hsmr TM-4 indeed contributes to the stability of SMR family dimers, an "interface switch" model of assembly emerges based on: (i) burial of the Gly face in computational models of the SMR monomer (36) and (ii) a proposed alteration in the SMR monomer fold that accompanies dimerization (5). In this model, shown in Fig. 10, the structural inequivalence of the two subunits in the SMR dimer (9–12) is precipitated by an interface switch, defined as a change at the Gly face in a single subunit from intramonomer contacts to intermonomer pairing with the Ala-Val face. Non-equivalent folds are therefore necessary in our model for assembly of the active asymmetric SMR dimer via two-faced TM-4/TM-4 contacts and also allow for, but do not require, symmetric self-association into a non-proliferating tetramer.

Although the applicability of our model of "interface switching" to SMR assembly remains to be tested in the full-length protein, structural studies imply that TM-4 has multiple interaction-competent surfaces (12) and that the helix-helix interactions in SMR family proteins can vary (11, 12). Any complete description of SMR assembly must also accommodate the TM-1/TM-1 contacts observed in EmrE (5), evaluate any contribution(s) of TM-2 and/or TM-3 to dimer stability, and test the effects of our mutations on the full-length protein. The oligomerization, assembly, and function of SMRs is nonetheless a complex process that must involve helix-helix interactions that can vary from monomer to monomer, a feature that may serve to impart the flexibility required for a protein pump and thus have implications for the SMR transport mechanism. Also, since oligomerization of SMR proteins is required for their drug efflux activity, our identification of TM-4 as a potential stabilizer of SMR dimers provides a foundation for design of novel therapeutics that target this multidrug resistance mechanism.


   FOOTNOTES
 
* This work was supported in part by a grant from the Canadian Institutes of HealthResearch (CIHR) (to C. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a post-doctoral award from the CIHR Training Program in Protein Folding:Principles and Diseases. Back

2 Recipient of a CIHR doctoral award. Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. Back

3 To whom correspondence should be addressed: Div. of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Tel.: 416-813-5924; Fax: 416-813-5005; E-mail: deber{at}sickkids.ca.

4 The 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. Back

5 A. Rath and C. M. Deber, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank S. Schuldiner for helpful discussion and comments on an earlier version of this manuscript.



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