Mutational analysis of tetracycline resistance protein transmembrane segment insertion

The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport. Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments. However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown. To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments. The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins. The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding. The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane. The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis.     

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.     

Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel

The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.     

The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator

The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.     

The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants

ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.     

Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.     

Membrane topology of the amino-terminal region of the sulfonylurea receptor.

The sulfonylurea receptor (SUR) is a member of the ATP-binding cassette family that is associated with Kir 6.x to form ATP-sensitive potassium channels. SUR is involved in nucleotide regulation of the channel and is the site of pharmacological interaction with sulfonylurea drugs and potassium channel openers. SUR contains three hydrophobic domains, TM(0), TM(1), and TM(2), with nucleotide binding folds following TM(1) and TM(2). Two topological models of SUR have been proposed containing either 13 transmembrane segments (in a 4+5+4 arrangement) or 17 transmembrane segments (in a 5+6+6 arrangement) (Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. t., Boyd, A. E., III, González, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423-426; Tusnády, G. E., Bakos, E., Váradi, A., and Sarkadi, B. (1997) FEBS Lett. 402, 1-3; Aguilar-Bryan, L., Clement, J. P., IV, González, G., Kunjilwar, K., Babenko, A., and Bryan, J. (1998) Physiol. Rev. 78, 227-245). We analyzed the topology of the amino-terminal TM(0) region of SUR1 using glycosylation and protease protection studies. Deglycosylation using peptide-N-glycosidase F and site-directed mutagenesis established that Asn(10), near the amino terminus, and Asn(1050) are the only sites of N-linked glycosylation, thus placing these sites on the extracellular side of the membrane. To study in detail the topology of SUR1, we constructed and expressed in vitro fusion proteins containing 1-5 hydrophobic segments of the TM(0) region fused to the reporter prolactin. The fusion proteins were subjected to a protease protection assay that reported the accessibility of the prolactin epitope. Our results indicate that the TM(0) region is comprised of 5 transmembrane segments. These data support the 5+6+6 model of SUR1 topology.     

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn168 in NS1 and Asn459 in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg368 and Arg87 respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn168/Arg368 and Asn459/Arg87 residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn168/Arg368 and Asn459/Arg87 pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.     

Identification of critical residues for transport activity of Acr3p,the Saccharomyces cerevisiae As(III)/H+ antiporter

Acr3p is an As(III)/H⁺ antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H⁺ exchange, we performed a systematic alanine‐replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H⁺ exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.     

Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6

Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me(2+) and H(+) into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd(2+) and H(+) uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp(34) couples H(+) and Me(2+) symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His(211) residue mediates pH-dependent Me(2+) uptake; MntH Asn(37), Asn(250), and Asn(401) in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp(34)-Pro-Gly (TMS1) and Met-Pro-His(211) (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H(+)-dependent Me(2+) import.     

Nucleotide binding, ATP hydrolysis, and mutation of the catalytic carboxylates of human P-glycoprotein cause distinct conformational changes in the transmembrane segments

P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.     

Disulfide cross-linking analysis shows that transmembrane segments 5 and 8 of human P-glycoprotein are close together on the cytoplasmic side of the membrane

Human P-glycoprotein (P-gp) transports a wide variety of structurally diverse compounds out of the cell. Knowledge about the packing of the transmembrane (TM) segments is essential for understanding the mechanism of drug recognition and transport. We used cysteine-scanning mutagenesis and disulfide cross-linking analysis to determine which TM segment in the COOH half of P-gp was close to TMs 5 and 6 since these segments in the NH(2) half are important for drug binding. An active Cys-less P-gp mutant cDNA was used to generate 240 double cysteine mutants that contained 1 cysteine in TMs 5 or 6 and another in TMs 7 or 8. The mutants were subjected to oxidative cross-linking analysis. No disulfide cross-linking was observed in the 140 TM6/TM7 or TM6/TM8 mutants. By contrast, cross-linking was detected in several P-gp TM5/TM8 mutants. At 4 degrees C, when thermal motion is low, P-gp mutants N296C(TM5)/G774C(TM8), I299C(TM5)/F770C(TM8), I299C(TM5)/G774C(TM8), and G300C(TM5)/F770C(TM8) showed extensive cross-linking with oxidant. These mutants retained drug-stimulated ATPase activity, but their activities were inhibited after treatment with oxidant. Similarly, disulfide cross-linking was inhibited by vanadate trapping of nucleotide. These results indicate that significant conformational changes must occur between TMs 5 and 8 during ATP hydrolysis. We revised the rotational symmetry model for TM packing based on our results and by comparison to the crystal structure of MsbA (Chang, G. (2003) J. Mol. Biol. 330, 419-430) such that TM5 is adjacent to TM8, TM2 is adjacent to TM11, and TMs 1 and 7 are next to TMs 6 and 12, respectively.     

Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) is quite promiscuous in that it can transport a broad range of structurally diverse compounds out of the cell. We hypothesized that the transmembrane (TM) segments that constitute the drug-binding site are quite mobile such that drug binding occurs through a "substrate-induced fit" mechanism. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for substrate-induced changes in the TM segments. Pairs of cysteines were introduced into a Cys-less P-gp and the mutants treated with oxidant (copper phenanthroline) in the presence or absence of various drug substrates. We show that cyclosporin A promoted cross-linking between residues P350C(TM6)/G939C(TM11), while colchicine and demecolcine promoted cross-linking between residues P350C(TM6)/V991C(TM12). Progesterone promoted cross-linking between residues P350C(TM6)/A935C(TM11), P350C(TM6)/G939C(TM11), as well as between residues P350C(TM6)/V991C(TM12). Other substrates such as vinblastine, verapamil, cis-(Z)-flupenthixol or trans-(E)-flupenthixol did not induce cross-linking at these sites. These results provide direct evidence that the packing of the TM segments in the drug-binding site is changed when P-gp binds to a particular substrate. The induced-fit mechanism explains how P-gp can accommodate a broad range of compounds.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

ATP hydrolysis promotes interactions between the extracellular ends of transmembrane segments 1 and 11 of human multidrug resistance P-glycoprotein

P-glycoprotein (P-gp, ABCB1) actively pumps a broad range of structurally unrelated cytotoxic compounds out of the cell. It has two homologous halves that are joined by a linker region. Each half has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD). Cross-linking studies have shown that the drug-binding pocket is at the interface between the TM domains. The two NBDs interact to form the ATP-binding sites. Coupling of ATP hydrolysis to drug efflux has been postulated to occur by conversion of the binding pocket from a high-affinity to a low-affinity state through alterations in the packing of the TM segments. TM 11 has also been reported to be important for drug binding. Here, we used cysteine-scanning mutagenesis and oxidative cross-linking to test for changes in the packing of TM 11 during ATP hydrolysis. We generated 350 double cysteine mutants that contained one cysteine at the extracellular end of TM11 and another cysteine at the extracellular ends of TMs 1, 3, 4, 5, or 6. The mutants were expressed in HEK293 cells and treated with oxidant in the absence or presence of ATP. Cross-linked product was not detected in SDS-PAGE gels in the absence of ATP. By contrast, cross-linked product was detected in mutants M68C(TM1)/Y950C(TM11), M68C(TM1)/Y953C(TM11), M68C(TM1)/A954C(TM11), M69C(TM1)/A954C(TM11), and M69C(TM1)/ F957C(TM11) in the presence of ATP but not with ADP or AMP.PNP. These results indicate that rearrangement of TM11 may contribute to the release of drug substrate during ATP hydrolysis.     

The pore-lining regions in cytochrome c oxidases: A computational analysis of caveolin,cholesterol and transmembrane helix contributions to proton movement

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain. CcO catalyzes a four electron reduction of O₂ to water at a catalytic site formed by high-spin heme (a₃) and copper atoms (Cu_B). While it is recognized that proton movement is coupled to oxygen reduction, the proton channel(s) have not been well defined. Using computational methods developed to study protein topology, membrane channels and 3D packing arrangements within transmembrane (TM) helix arrays, we find that subunit-1 (COX-1), subunit-2 (COX-2) and subunit-3 (COX-3) contribute 139, 46 and 25 residues, respectively, to channel formation between the mitochondrial matrix and intermembrane space. Nine of 12 TM helices in COX-1, both helices in COX-2 and 5 of the 6 TM helices in COX-3 are pore-lining regions (possible channel formers). Heme a₃ and the Cu_B sites (as well as the Cu_A center of COX-2) are located within the channel that includes TM-6, TM-7, TM-10 and TM-11 of COX-1 and are associated with multiple cholesterol and caveolin-binding (CB) motifs. Sequence analysis identifies five CB motifs within COX-1, two within COX-2 and four within COX-3; each caveolin containing a pore-lining helix C-terminal to a TM helix–turn–helix. Channel formation involves interaction between multiple pore-lining regions within protein subunits and/or dimers. PoreWalker analysis lends support to the D-channel model of proton translocation. Under physiological conditions, caveolins may introduce channel formers juxtaposed to those in COX-1, COX-2 and COX-3, which together with cholesterol may form channel(s) essential for proton translocation through the inner mitochondrial membrane.     

Expression of the tetA(C) tetracycline efflux pump in Escherichia coli confers osmotic sensitivity

Expression of tetA(C) in Escherichia coli confers resistance to tetracycline as well as sensitivity to nickel and cadmium salts, lipophilic chelating agents, and aminoglycoside antibiotics. In this report we determine that high-level expression of tetA(C) also confers an osmotic sensitivity. The osmotic-sensitive phenotype is distinct from the tetracycline-resistant phenotype and can be localized to a domain contained within the first 98 amino acid residues of the TetA(C) polypeptide.     

TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'. Subunit c' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.     

His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor

Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His⁴⁹⁹ near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His⁴⁹⁹ is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His⁴⁹⁹ regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.