Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Transmembrane interactions in the activation of the Neu receptor tyrosine kinase

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix-helix interactions stabilize the transmembrane dimer. We have determined the local secondary structure and interhelical contacts in the region of position 664 in peptide models of the activated receptor using solid-state rotational resonance and rotational echo double-resonance (REDOR) NMR methods. Intrahelical (13)C rotational resonance distance measurements were made between 1-(13)C-Thr662 and 2-(13)C-Gly665 on peptides corresponding to the wild-type Neu and activated Neu transmembrane sequences containing valine and glutamate at position 664, respectively. We observed similar internuclear distances (4.5 +/- 0.2 A) in both Neu and Neu*, indicating that the region near residue 664 is helical and is not influenced by mutation. Interhelical (15)N...(13)C REDOR measurements between Gln664 side chains on opposing helices were not consistent with hydrogen bonding between the side chain functional groups. However, interhelical rotational resonance measurements between 1-(13)C-Glu664 and 2-(13)C-Gly665 and between 1-(13)C-Gly665 and 2-(13)C-Gly665 demonstrated close contacts (4.3-4.5 A) consistent with the packing of Gly665 in the Neu* dimer interface. These measurements provide structural constraints for modeling the transmembrane dimer and define the rotational orientation of the transmembrane helices in the activated receptor.     

Comparison of proto-oncogenic and mutant forms of the transmembrane region of the Neu receptor in TFE

A single mutation within the transmembrane region of the Neu receptor (Val664-->Glu) is known to enhance tyrosine kinase activity, by promoting receptor dimerization. In order to gain insight into potential structural changes that arise as a result of the mutation, peptides corresponding to the complete transmembrane domain of proto-oncogenic and mutant forms of Neu have been studied by 1H nuclear magnetic resonance in the solvent trifluoroethanol (TFE). The chemical shifts are similar for both forms of the peptide, with the exception of amide residues close to the mutation site. Both peptides adopt a helical conformation, with a distinct bend one turn downstream of the mutation site. This deformation gives rise to several nuclear Overhauser effects, the majority of which were detected in both peptides, that are atypical for a straight canonical alpha-helix. Our data in this solvent do not support a conformational change in the transmembrane domain of monomeric Neu as a result of the mutation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that proto-oncogenic Neu peptides have a higher propensity to oligomerize in the solvent TFE than the Glu664 oncogenic form.     

Transmembrane helix packing of ErbB/Neu receptor in membrane environment: a molecular dynamics study

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains. Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the Neu(TM) helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization-competent structure responsible of the proper topology necessary for receptor activation.     

Transmembrane Helix Packing of ErbB/Neu Receptor in Membrane Environment: A Molecular Dynamics Study

Abstract

Dimerization or oligomerization of the ErbB/Neu receptors are necessary but not sufficient for initiation of receptor signaling. The two intracellular domains must be properly oriented for the juxtaposition of the kinase domains allowing trans-phosphorylation. This suggests that the transmembrane (TM) domain acts as a guide for defining the proper orientation of the intracellular domains.

Two structural models, with the two helices either in left-handed or in right-handed coiling have been proposed as the TM domain structure of the active receptor. Because experimental data do not distinguish clearly helix-helix packing, molecular dynamics (MD) simulations are used to investigate the energetic factors that drive Neu TM-TM interactions of the wild and the oncogenic receptor (Val664/Glu mutation) in DMPC or in POPC environments. MD results indicate that helix-lipid interactions in the bilayer core are extremely similar in the two environments and raise the role of the juxtamembrane residues in helix insertion and helix-helix packing. The TM domain shows a greater propensity to adopt a left-handed structure in DMPC, with helices in optimal position for strong inter-helical Hbonds induced by the Glu mutation. In POPC, the right-handed structure is preferentially formed with the participation of water in inter-helical Hbonds. The two structural arrangements of the NeuTM helices both with GG4 residue motif in close contact at the interface are permissible in the membrane environment. According to the hypothesis of a monomer-dimer equilibrium of the proteins it is likely that the bilayer imposes structural constraints that favor dimerization- competent structure responsible of the proper topology necessary for receptor activation.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.     

Characterization of the proto-oncogenic and mutant forms of the transmembrane region of Neu in micelles

We have investigated peptides corresponding to the complete transmembrane region of both proto-oncogenic (Val(664)) and mutant (Glu(664)) forms of the receptor Neu in detergent micelles by NMR and CD spectroscopy. Both forms of the peptide appear to adopt similar levels of helicity and dimeric interactions based on the analysis of CD spectra and nuclear Overhauser effect connectivity profiles. There are considerable differences in the chemical shifts of amide and, to a lesser extent, CHalpha resonances between the two forms of the peptides, and these differences are most pronounced in residues upstream of the mutation site and close to the N terminus of the transmembrane domain. Similarly, there are substantial differences in the amide hydrogen-deuterium exchange rates for residues close to and upstream of the mutation site; amide protons in this region of the protooncogenic peptide are much more resistant to exchange than those in the mutant form. In both molecules, residues downstream of the mutation site exhibit slow exchange. We therefore demonstrate that, although transmembrane Neu peptides exhibit similar levels of secondary structure when dispersed in detergent, there are detectable differences in their adopted micellar states that may provide insight into the dimer-promoting ability of the polar transforming mutation.     

Transmembrane peptides from tyrosine kinase receptor. Mutation-related behavior in a lipid bilayer investigated by molecular dynamics simulations

Polar mutations in transmembrane alpha helices may alter the structural details of the hydrophobic sequences and control intermolecular contacts. We have performed molecular dynamics simulations on the transmembrane domain of the proto-oncogenic and the oncogenic forms of the Neu receptor in a fluid DMPC bilayer to test whether the Glu mutation which replaces the Val residue at position 664 may alter the helical structure and its insertion in the membrane. The simulations show that the wild and the mutant forms of the transmembrane domain have a different behavior in the bilayer. The native transmembrane sequence is found to be more flexible than in the presence of the Glu mutation, characterized by a tendency to pi deformation to accommodate the helix length to the membrane thickness. The mutant form of this domain does not evidence helical deformation in the present simulation. Hydrophobic matching is achieved both by a larger helix tilt and a vertical shift of the helix towards the membrane interface, favoring the accessibility of the Glu side chain to the membrane environment. A rapid exchange of hydrogen bond interactions with the surrounding water molecules and the lipid headgroups is observed. The difference in the behavior between the two peptides in a membrane environment was also observed experimentally. Both simulation and experimental results agree with the hypothesis that water may act as an intermediate for the formation of cross links between the facing Glu side chains stabilizing the dimer.     

The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H > E > Q > K > G > V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.     

FGFR3 Transmembrane Domain Interactions Persist in the Presence of Its Extracellular Domain

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Förster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.     

Molecular dynamics simulations of the transmembrane domain of the oncogenic ErbB2 receptor dimer in a DMPC bilayer

Molecular dynamics simulations of an atomic model of the transmembrane domain of the oncogenic ErbB2 receptor dimer embedded in an explicit dimyristoylphosphatidylcholine (DMPC) bilayer were performed for more than 4 ns. The oncogenic Glu mutation in the membrane spanning segment plays a major role in tyrosine kinase activity and receptor dimerization, and is thought to be partly responsible for the structure of the transmembrane domain of the active receptor. MD results show that the interactions between the two transmembrane helices are characteristic of a left-handed packing as previously demonstrated from in vacuo simulations. Moreover, MD results reveal the absence of persistent hydrogen bonds between the Glu side chains in a membrane environment, which raise the question of the ability for Glu alone to stabilize the TM domain of the ErbB2 receptor. Interestingly the formation of the alpha-pi motif in the two ErbB2 transmembrane helices confirms the concept of intrinsic sequence-induced conformational flexibility. From a careful analysis of our MD results, we suggest that the left-handed helix-helix packing could be the key to correctly orient the intracellular domain of the activated receptor dimer. The prediction of such interactions from computer simulations represents a new step towards the understanding of signaling mechanisms.     

High-Throughput Selection of Transmembrane Sequences That Enhance Receptor Tyrosine Kinase Activation

Dimerization is a critical requirement for the activation of the intracellular kinase domains of receptor tyrosine kinases (RTKs). The single transmembrane (TM) helices of RTKs contribute to dimerization, but the details are not well understood. Work with TM helices in various model systems has revealed a small number of specific dimerization sequence motifs, and it has been suggested that RTK dimerization is modulated by such motifs. Yet questions remain about the universality of these sequence motifs for RTK dimerization and about how TM domain dimerization in model systems relates to RTK activation in mammalian membranes. To investigate these questions, we designed a 3888-member combinatorial peptide library based on the TM domain of Neu (ErbB2) as a model RTK. The library contains many closely related, Neu-like sequences, including thousands of sequences with known dimerization motifs. We used an SDS-PAGE-based screen to select peptides that dimerize better than the native Neu sequence, and we assayed the activation of chimeric Neu receptors in mammalian cells with TM sequences selected in the screen. Despite the very high abundance of known dimerization motifs in the library, only a very few dimerizing sequences were identified by SDS-PAGE. About half of those sequences activated the Neu kinase significantly more than did the wild-type TM sequence. This work furthers our knowledge about the requirements for membrane protein interactions and the requirements for RTK activation in cells.     

Biochemical comparisons of the normal and oncogenic forms of insect cell-expressed neu tyrosine kinases.

The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.     

Atomistic mechanism of the constitutive activation of PDGFRA via its transmembrane domain

Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA.     

A subdomain in the transmembrane domain is necessary for p185neu* activation.

The neu proto-oncogene encodes a protein highly homologous to the epidermal growth factor receptor. The neu protein (p185) has a molecular weight of 185,000 Daltons and, like the EGF receptor, possesses tyrosine kinase activity. neu is activated in chemically induced rat neuro/glioblastomas by substitution of valine 664 with glutamic acid within the transmembrane domain. The activated neu* protein (p185*) has an elevated tyrosine kinase activity and a higher propensity to dimerize, but the mechanism of this activation is still unknown. We have used site-directed mutagenesis to explore the role of specific amino acids within the transmembrane domain in this activation. We found that the lateral position and rotational orientation of the glutamic acid in the transmembrane domain does not correlate with transformation. However, the primary structure in the vicinity of Glu664 plays a significant role in this activation. Our results suggest that the Glu664 activation involves highly specific interactions in the transmembrane domain of p185.     

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.     

Molecular dynamics (MD) investigations of preformed structures of the transmembrane domain of the oncogenic Neu receptor dimer in a DMPC bilayer

The critical Val/Glu mutation in the membrane spanning domain of the rat Neu receptor confers the ability for ligand-independent signaling and leads to increased dimerization and transforming ability. There is evidence that the two transmembrane interacting helices play a role in receptor activation by imposing orientation constraints to the intracellular tyrosine kinase domains. By using MD simulations we have attempted to discriminate between correct and improper helix-helix packing by examining the structural and energetic properties of preformed left-handed and right-handed structures in a fully hydrated DMPC bilayer. The best energetic balance between the residues at the helix-helix interface and the residues exposed to the lipids is obtained for helices in symmetrical left-handed interactions packed together via Glu side chain/Ala backbone interhelical hydrogen bonds. Analyses demonstrate the importance of the ATVEG motif in helix-helix packing and point to additional contacting residues necessary for association. Our findings, all consistent with experimental data, suggest that a symmetrical left-handed structure of the helices could be the transmembrane domain configuration that promotes receptor activation and transformation. The present study may provide further insight into signal transduction mechanisms of the ErbB/Neu receptors.     

In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain

The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes cell transformation by binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). In order to productively interact with the receptor, it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers, and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer; indeed, mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable alpha-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation demonstrate for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol (-1). We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function.     

The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C‐terminal juxtamembrane residues

The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of α and β integrin TM interactions. However, we show herein that in FGFR3‐TM, four C‐terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3‐TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C‐terminal residues were present. In the absence of these four residues, A391E was dimer‐destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3‐TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR‐TM.