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.     

Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (K_d = 7.2 ± 0.4 μM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (ΔG = −15.4 ± 0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.     

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.     

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.     

Structural characterization of a dimerization interface in the CD28 transmembrane domain

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The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Effects of the oncogenic V(664)E mutation on membrane insertion, structure, and sequence-dependent interactions of the Neu transmembrane domain in micelles and model membranes: an integrated biophysical and simulation study

Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V(664)E) mutation at the center of its α-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change in behavior upon introduction of the V(664)E mutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V(664)E mutation in the isolated TM domain with respect to protein-protein and protein-lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V(664)E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed.     

The transmembrane domain of Neu in a lipid bilayer: molecular dynamics simulations

The results of full-atom molecular dynamics simulations of the transmembrane domains (TMDs) of both native, and Glu⁶⁶⁴-mutant (either protonated or unprotonated) Neu in an explicit fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer are presented. For the native TMD peptide, a 10.05 ns trajectory was collected, while for the mutant TMD peptides 5.05 ns trajectories were collected for each. The peptides in all three simulations display stable predominantly -helical hydrogen bonding throughout the trajectories. The only significant exception occurs near the C-terminal end of the native and unprotonated mutant TMDs just outside the level of the lipid headgroups, where -helical hydrogen bonding develops, introducing a kink in the backbone structure. However, there is no indication of the formation of a bulge within the hydrophobic region of either native or mutant peptides. Over the course of the simulation of the mutant peptide, it is found that a significant number of water molecules penetrate the hydrophobic region of the surrounding lipid molecules, effectively hydrating Glu⁶⁶⁴. If the energy cost of such water penetration is significant enough, this may be a factor in the enhanced dimerization affinity of Glu⁶⁶⁴-mutant Neu.     

The tumor suppressor RECK interferes with HER-2/Neu dimerization and attenuates its oncogenic signaling

Our previous study demonstrates that HER-2/Neu oncogene inhibits a matrix metalloproteinase inhibitor and tumor metastasis suppressor RECK to promote metastasis. Conversely, the effect of RECK on the oncogenic function of HER-2/Neu is unknown. Ectopic expression of RECK in 293T cells and HER-2/Neu-overexpressing breast cancer cells shows that RECK and HER-2/Neu are co-localized and these two proteins can be co-immunoprecipitated. RECK inhibits HER-2/Neu receptor dimerization and autophosphorylation, which causes reduction of ERK and AKT kinase activity and down-regulation of HER-2/Neu target genes. RECK expression is reduced in 58.8% of breast cancer tissues and is associated with lymph node invasion supporting its anti-metastatic role. Collectively, we provide the first evidence that RECK can negatively regulate oncogenic activity of HER-2/Neu by inhibiting receptor dimerization.

Structured summary

HER-2/Neuphysically interacts with HER-2/Neu by blue native page (View interaction)HER-2/Neuphysically interacts with RECK by coimmunoprecipitation (View interaction)HER-2/Neu and RECKcolocalize by fluorescence microscopy (View Interaction 1, 2)HER-2/Neuphysically interacts with RECK by anti bait coimmunoprecipitation (View interaction)     

Dimerization of Neu/Erb2 transmembrane domain is controlled by membrane curvature

Secondary structures of the proto-oncogenic Neu/ErbB2 transmembrane segment and its mutant analogue have been determined in phospholipids. It is found that the mutated peptide possesses less helical character possibly due to the valine/glutamic acid point mutation. Embedding peptides in lipid systems whose topology can change from small (100-200 A) tumbling objects to bilayer discs of 450 A diameter leads to the finding that coiled-coil interactions are only observed in the presence of a bilayer membrane of low curvature, independent of mutation. This strongly suggests that any event that may change membrane topology can therefore perturb the dimerization/ologomerization and subsequent phosphorylation cascade leading to cell growth or cancer processes.     

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor,a human GPCR

The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His)₆ tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.     

Synthesis and conformational studies of a transmembrane domain from a diverged microsomal Delta(12)-desaturase

Transmembrane domains of the acyl-coenzyme A and acyl phosphatidylcholine-utilizing desaturases may control interactions with electron transport domains, be involved in substrate specificity and/or serve as a structural foundation for the enzyme. To experimentally define these domains and as a prelude to detailed NMR studies, a segment of the microsomal Delta(12)-desaturase/acetylenase CREP-1 predicted to contain the amino-proximate transmembrane domain TM-A was chemically synthesized. A modified 9-fluorenylmethoxycarbonyl procedure was used that ensured complete deprotections at each homologation and the peptide was purified in good yield by reverse-phase high-performance liquid chromatography. Conformational studies of the hydrophobic peptide TM-A demonstrated its strong propensity for folding into an alpha-helical secondary structure. The helical content was 58-65% in aqueous solutions containing 40-80% 2,2,2-trifluoroethanol, a lipomimetic solvent, and was maximal at low temperatures. The peptide assumed a largely helical character when incorporated into phospholipid bilayers and detergent micelles. Experimental evidence is in agreement with neural network predictions that a transmembrane domain exists between residues R-44 and I-67 in this diverged Delta(12)-desaturase.     

Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR

Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning α-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity.     

Structure prediction of the dimeric neu/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations

Dimerization of the neu/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of the helical transmembrane domain (TM) of neu and ErbB-2 receptors are used to predict their dimer structure both in the wild and oncogenic forms. A global conformational search method, applied to define the best orientations of parallel helices, showed an energetically favorable configuration with the specific mutation site within the interface, common for both the nontransforming and the transforming neu/ErbB-2 TM dimers. Starting from this configuration, a total of 10 simulations, about 1.4 ns each, performed in vacuum, without any constraints, show that the two helices preferentially wrap in left-handed interactions with a packing angle at about 20°. The resulting structures are nonsymmetric and the hydrogen bond network analysis shows that helices experience π local distortions that facilitate inter-helix hydrogen bond interactions and may result in a change in the helix packing, leading to a symmetric interface. For the mutated sequences, we show that the Glu side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. We show that the connectivity between interfacial residues conforms to the knobs-into-holes packing mode of transmembrane helices. The dimeric interface described in our models is discussed with respect to mutagenesis studies. Received: 12 March 1999 / Revised version: 23 August 1999 / Accepted: 23 August 1999     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.