Active conformation of the erythropoietin receptor: random and cysteine-scanning mutagenesis of the extracellular juxtamembrane and transmembrane domains

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.     

Active and inactive orientations of the transmembrane and cytosolic domains of the erythropoietin receptor dimer

Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.     

Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function

The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.     

Insights into the transmembrane helix associations of kit ligand by molecular dynamics simulation and TOXCAT

Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self‐associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362–1370. © 2017 Wiley Periodicals, Inc.     

Receptor tyrosine kinase transmembrane domains: Function,dimer structure and dimerization energetics

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) play an active role in signaling. They contribute to the stability of full-length receptor dimers and to maintaining a signaling-competent dimeric receptor conformation. In an exciting new development, two structures of RTK TM domains have been solved, a break-through achievement in the field. Here we review these structures, and we discuss recent studies of RTK TM domain dimerization energetics, possible synergies between domains, and the effects of pathogenic RTK TM mutations on structure and dimerization.Key words: transmembrane domain, dimerization thermodynamics, receptor tyrosine kinases, pathogenic mutations, dimer structure     

Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between (835)Ala and (918)Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.     

Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations

Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin’s TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes.     

The erythropoietin receptor transmembrane domain mediates complex formation with viral anemic and polycythemic gp55 proteins

Erythropoietin receptor (EpoR) activation is crucial for mature red blood cell production. The murine EpoR can also be activated by the envelope protein of the polycythemic (P) spleen focus forming virus (SFFV), gp55-P. Due to differences in the TM sequence, gp55 of the anemic (A) strain SFFV, gp55-A, cannot efficiently activate the EpoR. Using antibody-mediated immunofluorescence co-patching, we show that the majority of EpoR forms hetero-oligomers at the cell surface with gp55-P and, surprisingly, with gp55-A. The EpoR TM domain is targeted by gp55-P and -A, as only chimeric receptors containing EpoR TM sequences oligomerized with gp55 proteins. Both gp55-P and gp55-A are homodimers on the cell surface, as shown by co-patching. However, when the homomeric interactions of the isolated TM domains were assayed by TOXCAT bacterial reporter system, only the TM sequence of gp55-P was dimerized. Thus, homo-oligomerization of gp55 proteins is insufficient for full EpoR activation, and a correct conformation of the dimer in the TM region is required. This is supported by the failure of gp55-A-->P, a mutant protein whose TM domain can homo-oligomerize, to fully activate EpoR. As unliganded EpoR forms TM-dependent but inactive homodimers, we propose that the EpoR can be activated to different extents by homodimeric gp55 proteins, depending on the conformation of the gp55 protein dimer in the TM region.     

Molecular dynamics simulation approach for the prediction of transmembrane helix–helix heterodimers assembly

Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor. The method successfully reproduces the experimental structures of the TM domain of glycophorin A (GpA(TM)) with a root mean square deviation of 1.5 A. The search protocol identifies three energetically stable models of the TM domain of HER2-HER3 receptor with favorable helix-helix arrangement, including right-handed and left-handed coiled-coils. The predicted TM structures exhibit the GxxxG-like motif at the dimer interface which is presumed to drive receptor oligomerization. We demonstrate that native structures of TM domain can be predicted without quantitative experimental data. This search protocol could help to predict structures of the TM domain of HER heterodimer family.     

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.     

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.     

Making structural sense of dimerization interfaces of delta opioid receptor homodimers

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.     

Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.     

Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor

Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor-like conformation precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR), the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240-Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR(-/-) mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.     

Ligand sensitivity in dimeric associations of the serotonin 5HT2c receptor

G-protein-coupled receptors (GPCRs) respond to external stimuli by activating heterotrimeric G proteins inside the cell. There is increasing evidence that many GPCRs exist as dimers or higher oligomers, but the biochemical nature of such dimers and what roles they have, if any, in signal transduction remains unclear. We conducted a comprehensive study of dimerization of the 5HT2c serotonin receptor using disulphide-trapping experiments. We found a dimer interface between transmembrane (TM) helices IV and V that is markedly sensitive to the state of receptor activation. This dimer seems to be quasisymmetrical in interfacial geometry and asymmetrical in its association with its cognate G alpha protein. We also found a second interface at TM I helices, which is insensitive to the state of activation.     

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

Hepatitis C virus (HCV) NS4A is a single-pass transmembrane (TM) protein essential for viral replication and particle assembly. The sequence of the NS4A TM domain is highly conserved, suggesting that it may be important for protein-protein interactions. To test this hypothesis, we measured the potential dimerization of the NS4A TM domain in a well-characterized two-hybrid TM protein interaction system. The NS4A TM domain exhibited a strong homotypic interaction that was comparable in affinity to glycophorin A, a well-studied human blood group antigen that forms TM homodimers. Several mutations predicted to cluster on a common surface of the NS4A TM helix caused significant reductions in dimerization, suggesting that these residues form an interface for NS4A dimerization. Mutations in the NS4A TM domain were further examined in the JFH-1 genotype 2a replicon system; importantly, all mutations that destabilized NS4A dimers also caused defects in RNA replication and/or virus assembly. Computational modeling of NS4A TM interactions suggests a right-handed dimeric interaction of helices with an interface that is consistent with the mutational effects. Furthermore, defects in NS4A oligomerization and virus particle assembly of two mutants were rescued by NS4A A15S, a TM mutation recently identified through forward genetics as a cell culture-adaptive mutation. Together, these data provide the first example of a functionally important TM dimer interface within an HCV nonstructural protein and reveal a fundamental role of the NS4A TM domain in coordinating HCV RNA replication and virus particle assembly.     

Packing density of the erythropoietin receptor transmembrane domain correlates with amplification of biological responses

The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.     

A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis

The "eukaryotic-like" receptor Ser/Thr protein kinases (STPKs) are candidates for the sensors that mediate environmental adaptations of Mycobacterium tuberculosis (Mtb). To define the mechanisms of regulation and substrate recognition, we determined the crystal structure of the ligand-free, activated kinase domain (KD) of the Mtb STPK, PknE. Remarkably, the PknE KD formed a dimer similar to that first observed in the structure of the ATPgammaS complex of the Mtb paralog, PknB. This structural similarity, which occurs despite little sequence conservation between the PknB and PknE dimer interfaces, supports the idea that dimerization regulates the Mtb receptor STPKs. Insertion of the DFG motif into the ATP-binding site and other conformational differences compared the ATPgammaS:PknB complex suggest that apo-PknE is not pre-organized to bind nucleotides. This structure may represent an inactive conformation stabilized by dimerization or, alternatively, an active conformation that reveals shifts that mediate nucleotide exchange and order substrate binding.