The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.     

Inversion of thermosensing property of the bacterial receptor Tar by mutations in the second transmembrane region

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.     

Tuning a bacterial chemoreceptor with protein-membrane interactions

Chemoreceptors in Escherichia coli are homodimeric transmembrane proteins that convert environmental stimuli into intracellular signals controlling flagellar motion. Chemoeffectors bind to the extracellular (periplasmic) domain of the receptors, whereas their cytoplasmic domain mediates signaling and adaptation. The second transmembrane helix (TM2) connects these two domains. TM2 contains an aliphatic core flanked by amphipathic aromatic residues that have specific affinity for polar-hydrophobic membrane interfaces. We previously showed that Trp-209, near the cytoplasmic end of TM2, helps maintain the normal baseline-signaling state of the aspartate chemoreceptor (Tar) and that Tyr-210 plays an auxiliary role in this control. We have now repositioned the Trp-209/Tyr-210 pair in single-residue increments about the cytoplasmic polar-hydrophobic interface. Changes from WY-2 to WY+1 modulate the baseline-signaling state of the receptor in predictable and incremental steps that can be compensated by adaptive methylation/demethylation. Greater displacements, as in WY-3, WY+2, and WY+3, bias the receptor to the off kinase-inhibiting state or the on kinase-stimulating state, respectively, to a degree that cannot be fully compensated by the adaptation system. Aromatic residues analogous to Trp-209/Tyr-210 are present in other chemoreceptors and many transmembrane sensor kinases, where they may serve a similar function.     

Phenol sensing by Escherichia coli chemoreceptors: a nonclassical mechanism

The four transmembrane chemoreceptors of Escherichia coli sense phenol as either an attractant (Tar) or a repellent (Tap, Trg, and Tsr). In this study, we investigated the Tar determinants that mediate its attractant response to phenol and the Tsr determinants that mediate its repellent response to phenol. Tar molecules with lesions in the aspartate-binding pocket of the periplasmic domain, with a foreign periplasmic domain (from Tsr or from several Pseudomonas chemoreceptors), or lacking nearly the entire periplasmic domain still mediated attractant responses to phenol. Similarly, Tar molecules with the cytoplasmic methylation and kinase control domains of Tsr still sensed phenol as an attractant. Additional hybrid receptors with signaling elements from both Tar and Tsr indicated that the transmembrane (TM) helices and HAMP domain determined the sign of the phenol-sensing response. Several amino acid replacements in the HAMP domain of Tsr, particularly attractant-mimic signaling lesions at residue E248, converted Tsr to an attractant sensor of phenol. These findings suggest that phenol may elicit chemotactic responses by diffusing into the cytoplasmic membrane and perturbing the structural stability or position of the TM bundle helices, in conjunction with structural input from the HAMP domain. We conclude that behavioral responses to phenol, and perhaps to temperature, cytoplasmic pH, and glycerol, as well, occur through a general sensing mechanism in chemoreceptors that detects changes in the structural stability or dynamic behavior of a receptor signaling element. The structurally sensitive target for phenol is probably the TM bundle, but other behaviors could target other receptor elements.     

Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein,Tez1

Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker. Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype. Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction. Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype. Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response. None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function. In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation. Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input. In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.     

Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues

Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.     

Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V(1a) vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.     

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Functional role of tryptophan residues in the fourth transmembrane domain of the CB(2) cannabinoid receptor

Several tryptophan (Trp) residues are conserved in G protein-coupled receptors (GPCRs). Relatively little is known about the contribution of these residues and especially of those in the fourth transmembrane domain in the function of the CB(2) cannabinoid receptor. Replacing W158 (very highly conserved in GPCRs) and W172 (conserved in CB(1) and CB(2) cannabinoid receptors but not in many other GPCRs) of the human CB(2) receptor with A or L or with F or Y produced different results. We found that the conservative change of W172 to F or Y retained cannabinoid binding and downstream signaling (inhibition of adenylyl cyclase), whereas removal of the aromatic side chain by mutating W172 to A or L eliminated agonist binding. W158 was even more sensitive to being mutated. We found that the conservative W158F mutation retained wild-type binding and signaling activities. However, W158Y and W158A mutants completely lost ligand binding capacity. Thus, the Trp side chains at positions 158 and 172 seem to have a critical, but different, role in cannabinoid binding to the human CB(2) receptor.     

Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor

Biophysical study of the transmembrane (TM) domains of integral membrane proteins has traditionally been impeded by their hydrophobic nature. As a result, an understanding of the details of protein-protein interactions within membranes is often lacking. We have demonstrated previously that model TM segments with flanking cationic residues spontaneously fold into alpha-helices upon insertion into membrane-mimetic environments. Here, we extend these studies to investigate whether such constructs consisting of TM helices from biological systems retain their native secondary structures and oligomeric states. Single-spanning TM domains from the epidermal growth factor receptor (EGFR), glycophorin A (GPA), and the influenza A virus M2 ion channel (M2) were designed and synthesized with three to four lysine residues at both N- and C-termini. Each construct was shown to adopt an alpha-helical conformation upon insertion into sodium dodecyl sulfate micelles. Furthermore, micelle-inserted TM segments associated on SDS-PAGE gels according to their respective native-like oligomeric states: EGFR was monomeric, GPA was dimeric, and M2 was tetrameric. This approach was then used to investigate whether one or both of the TM segments (Tar-1 and Tar-2) from the Escherichia coli aspartate receptor were responsible for its homodimeric nature. Our results showed that Tar-1 formed SDS-resistant homodimers, while Tar-2 was monomeric. Furthermore, no heterooligomerization between Tar-1 and Tar-2 was detected, implicating the Tar-1 helix as the oligomeric determinant for the Tar protein. The overall results indicate that this approach can be used to elucidate the details of TM domain folding for both single-spanning and multispanning membrane proteins.     

The composition rather than position of polar residues (QxxS) drives aspartate receptor transmembrane domain dimerization in vivo

Transmembrane (TM) helix association is an important process affecting the function of many integral membrane proteins. Consequently, aberrations in this process are associated with diseases. Unfortunately, our knowledge of the factors that control this oligomerization process in the membrane milieu is limited at best. Previous studies have shown a role for polar residues in the assembly of synthetic peptides in vitro and the association of de novo-designed TM helices in vivo. Here we examined, for the first time, the involvement of polar residues in the dimerization of a biological TM domain in its natural environment. We analyzed both the involvement of polar residues in the dimerization process and whether their influence is position-dependent. For this purpose, we used the TM domain of the Escherichia coli aspartate receptor (Tar) and 10 single and double mutants. Polar to nonpolar mutations in the sequence demonstrated the role of the QxxS motif in the dimerization of the Tar TM domain. Moreover, creating a GxxxG motif, instead of the polar motif, almost completely abolished dimerization. Swapping positions between two wild-type polar residues did not affect dimerization, implying a similar contribution from both positions. Interestingly, mutants that contain two identical strong polar residues, EE and QQ, demonstrated a substantially higher level of dimerization than a QE mutant, although all three TM domains contain two strong polar residues. This result suggests that, in addition to the polarity of the residues, the formation of symmetric bonds also plays a role in dimer stability. The results of this study may facilitate a rational modulation of membrane protein function for therapeutic purposes.     

Ligand specificity determined by differentially arranged common ligand-binding residues in bacterial amino acid chemoreceptors Tsr and Tar

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.     

The role of individual amino acids in the dimerization of CR4 and ACR4 transmembrane domains

CRINKLY4 is a growth factor-like plant receptor kinase designated as CR4 in Zea mays and ACR4 in Arabidopsis. Using the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment, we have previously demonstrated that the dimerization potential of the ACR4 transmembrane (TM) domain is significantly weaker than that of CR4 TM domain, even though 13 of the 24 residues are identical. Neither of the TM domains contain the GxxxG motif that has been shown to be important for the dimerization of the TM segments of several receptors. To further investigate the relationship between protein sequence and dimerization potential, we (a) mutated each of the 11 differing residues in the CR4 TM domain to the corresponding residue of ACR4 (b) made reciprocal mutations in ACR4 and (c) made hybrids consisting of half CR4 and half ACR4 TM domains. Our results suggest that most mutations in ACR4 or CR4 TM domains have low to moderate effects on the dimerization potential and that residues in the N-terminal half of the CR4 TM domain are important for dimerization.     

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.     

Role of threonine residue 154 in ligand recognition of the tar chemoreceptor in Escherichia coli.

The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate, maltose, and phenol, repellent responses to Ni2+ and Co2+, and thermoresponses. To understand the role of threonine residue 154, which is located in the ligand-binding domain of Tar, we replaced the residue with serine, isoleucine, and proline by site-directed mutagenesis. The replacements caused reductions in aspartate sensing but had only a small effect on maltose sensing and almost no effect on phenol sensing, repellent sensing, and thermosensing. These results indicate that Thr-154 of Tar is rather specifically involved in aspartate sensing. The reductions in the response threshold for aspartate by the replacements with serine, isoleucine, and proline were less than 1, about 2, and more than 5 orders of magnitude, respectively. When the corresponding threonine residue in the Tsr chemoreceptor was replaced with the same amino acids, roughly similar reductions in the response threshold for serine resulted. Thus, these threonine residues seem to have a common role in detecting the aspartate and serine attractant families. A mechanism by which these chemoreceptors detect the amino acid attractants is discussed.     

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.     

Two motifs within a transmembrane domain, one for homodimerization and the other for heterodimerization

Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the N-terminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.     

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.