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Spatial Structure of the Dimeric Transmembrane Domain of the Growth Factor Receptor ErbB2 Presumably Corresponding to the Receptor Active State

2008; Elsevier BV; Volume: 283; Issue: 11 Linguagem: Inglês

10.1074/jbc.m709202200

1083-351X

Eduard V. Bocharov, Константин С. Минеев, Pavel E. Volynsky, Yaroslav S. Ermolyuk, Elena N. Tkach, Alexander G. Sobol, Vladimir Chupin, М. П. Кирпичников, Roman G. Efremov, Alexander S. Arseniev,

Monoclonal and Polyclonal Antibodies Research

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed α-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations. Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed α-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations. The epidermal growth factor receptor (or ErbB) family is an important class of receptor tyrosine kinases involved in transmission of biochemical signals governing cell fate (1Schlessinger J. Cell. 2000; 281: 211-225Abstract Full Text Full Text PDF Scopus (3556) Google Scholar). Four human ErbB family members form numerous homo- and heterodimer combinations and bind different epidermal growth factor-related ligands, thus performing diverse functions in a complex signaling network (2Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). The binding of peptide growth factors to the extracellular domain of the receptor triggers the dimerization of receptor monomers or a change in the relative orientation of monomers in preformed receptor dimers, leading to autophosphorylation of tyrosine residues in the cytoplasmic kinase domain (3Tanner K.G. Kyte J. J. Biol. Chem. 1999; 274: 35985-35990Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar). Biochemical and genetic studies have revealed that the single-helix transmembrane (TM) 3The abbreviations used are:TMtransmembraneMDmolecular dynamicsDMPCdimyristoylphosphatidylcholineDHPCdihexanoylphosphatidylcholineNOEnuclear Overhauser effectNOESYNOE spectroscopy. domains of ErbB play an active role in the dimerization process and associate strongly in the absence of extracellular ligand-binding and cytoplasmic kinase domains (5Bennasroune A. Fickova M. Gardin A. Dirrig-Grosch S. Aunis D. Cremel G. Hubert P. Mol. Biol. Cell. 2004; 15: 3464-3474Crossref PubMed Scopus (101) Google Scholar, 6Duneau J.-P. Vegh A.P. Sturgis J.N. Biochemistry. 2007; 46: 2010-2019Crossref PubMed Scopus (69) Google Scholar). Mutational analysis assumed that the dimerization involves consensus small-X3-small (so-called GG4-like) motifs, formed by residues with small side chains allowing tight helix packing (7Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 8Curran A.R. Engelmann D.M. Curr. Opin. Struct. Biol. 2003; 13: 412-417Crossref PubMed Scopus (200) Google Scholar, 9Mackenzie K.R. Chem. Rev. 2006; 106: 1931-1977Crossref PubMed Scopus (183) Google Scholar). Receptor tyrosine kinase TM sequences often contain several remote GG4-like motifs, suggesting the ability of their TM domains to adopt more than one conformation, e.g. upon so-called rotation-coupled activation of the receptor (4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar, 10Jiang G. Hunter T. Curr. Biol. 1999; 9: 568-571Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar). Recent molecular modeling and solid-state NMR studies performed to predict the spatial structures of the dimeric TM domains of the human ErbB2 receptor and its rat homolog have disclosed two possible dimer conformations with interfaces located at either the N or C terminus of the α-helical TM segment, employing different GG4-like motifs for dimerization (7Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar, 12Enosh A.R. Fleishman S.J. Ben-Tal N. Halperin D. Bioinformatics (Oxf.). 2006; 23: e212-e218Crossref Scopus (17) Google Scholar, 13Smith S.O. Smith C. Shekar S. Peersen O. Ziliox M. Aimoto S. Biochemistry. 2002; 41: 9321-9332Crossref PubMed Scopus (71) Google Scholar). Nevertheless, an experimental spatial structure of the dimeric TM domain for ErbB2 as well as for any other receptor tyrosine kinase family members has not been reported so far. transmembrane molecular dynamics dimyristoylphosphatidylcholine dihexanoylphosphatidylcholine nuclear Overhauser effect NOE spectroscopy. Here, we present the high resolution structure of the homodimeric ErbB2 TM domain in a membrane-mimicking lipid environment solved by a heteronuclear NMR technique combined with molecular dynamics (MD) relaxation in an explicit membrane. Our results distinguish one of the potential conformations of the homodimer, which can be ascribed to the active state of the tyrosine kinase. On the basis of the analysis of the local conformation of the dimerization interface, we propose a molecular mechanism of action of some ErbB2 pro-oncogenic mutations, assuming overstabilization of the described TM domain conformation. Protein Expression and Purification—The DNA sequence encoding ErbB2 TM fragment 641–684 (referred to as ErbB2tm) was synthesized from six oligonucleotides by PCR. The TrxA-ErbB2tm fusion protein was constructed by fusing the gene for thioredoxin from Escherichia coli with an N-terminal His tag extension to the gene for ErbB2tm in the pGEMEX1 vector (Promega). To facilitate purification, the His tag and enterokinase cleavage site were placed between the genes for thioredoxin and ErbB2tm. The fusion protein was expressed in E. coli BL21(DE3) pLysS cells and grown in 2-liter flasks at 37 °C in M9 minimal medium containing (15NH4)2SO4 or (15NH4)2SO4/[U-13C]glucose (both from Cambridge Isotope Laboratories, Inc.) for the production of uniformly 15N- or 15N/13C-labeled protein samples. After induction with 50 μm isopropyl β-d-thiogalactopyranoside and an additional 40 h of growth at 13 °C, the cells were harvested and stored at -20 °C. Cell pellets (1-liter equivalents) were resuspended in 50 ml of lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 10 mm 2-mercaptoethanol, 10 mm imidazole, 15 mm Triton X-100, and 0.2 mm phenylmethylsulfonyl fluoride) and lysed by ultrasonication. Centrifugally clarified lysate was applied to Chelating Sepharose Fast Flow beads (Amersham Biosciences) pretreated with NiSO4 and eluted with 200 mm imidazole. After overnight incubation with the recombinant human enterokinase light chain, the cleaved ErbB2tm fragment was passed through Chelating Sepharose Fast Flow, loaded onto an SP-Sepharose Fast Flow column (Amersham Biosciences), and eluted with a gradient of NaCl. Yields of ∼10 mg of protein/liter of cell culture could be obtained by this procedure. Protein identity and purity were confirmed by gel electrophoresis, mass spectrometry, and NMR spectroscopy in a 1:1 methanol/chloroform mixture containing 5–10% water. NMR Spectroscopy and Structure Calculations—NMR experiments were performed using a 600-MHz (1H) Varian UNITY spectrometer equipped with a pulsed-field gradient unit and a triple-resonance probe. NMR spectra were acquired at 40 °C using 1 mm samples of ErbB2tm incorporated into 1:4 dimyristoylphosphatidylcholine (DMPC)/dihexanoylphosphatidylcholine (DHPC) lipid bicelles (at a lipid/protein molar ratio of 35) dissolved in buffer (pH 5.0) containing 20 mm deuterated sodium acetate, 6 mm dithiothreitol, 0.15 μm sodium azide, 1 mm EDTA, and either 5 or 99.9% D2O unless specified otherwise. Three ErbB2tm samples were prepared: uniformly 15N/13C-labeled, 15N-labeled, and a 1:1 mixture of uniformly 15N/13C-labeled and unlabeled proteins ("heterodimer" sample). The samples were initially subjected to several freeze/thaw cycles, resulting in uniform protein distribution among the lipid bicelles. To verify the validity of the NMR experimental conditions, dynamic light scattering and circular dichroism studies of ErbB2tm solubilized with the DMPC/DHPC bicelles and DMPC unilamellar liposomes (phospholipid bilayer) were performed (see supplemental "Experimental Procedures" and supplemental Fig. S1). The backbone and side chain 1H, 13C, and 15N resonances of ErbB2tm were assigned using standard triple-resonance techniques (14Sattler M. Schleucher J. Griesinger C. Prog. NMR Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1398) Google Scholar, 15Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. 2nd Ed. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego, CA2006Google Scholar). Two- and three-dimensional 1H-15N (Fig. 1A) and 1H-13C heteronuclear single quantum coherence, 15N-edited total correlation (40-ms mixing time), HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH spectra in H2O provided backbone and partial side chain assignments, whereas HCCH total correlation spectroscopy (15.6- and 23.4-ms mixing times) and 1H homonuclear nuclear Overhauser effect (NOE) spectroscopy (NOESY; 60-ms mixing time) experiments in D2O facilitated side chain assignments. Resonance assignments were performed with CARA software. The value of the heteronuclear 15N{1H} steady-state NOE and the 15N longitudinal (T1) and transverse (T2) relaxation times were obtained for the 15N-labeled sample as described (16Bocharov E.V. Sobol A.G. Pavlov K.V. Korzhnev D.M. Jaravine V.A. Gudkov A.T. Arseniev A.S. J. Biol. Chem. 2004; 279: 17697-17706Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The effective rotation correlation times (τR) for the individual 15N nuclei were calculated from the T1/T2 ratio using DASHA (17Orekhov V.Y. Nolde D.E. Golovanov A.P. Korzhnev D.M. Arseniev A.S. Appl. Magn. Reson. 1995; 9: 581-588Crossref Scopus (80) Google Scholar). The effective molecular mass of the ErbB2tm dimer embedded into the bicelle was estimated according to the empirical dependence (18Daragan V.A. Mayo K.H. Prog. NMR Spectrosc. 1997; 31: 63-105Abstract Full Text Full Text PDF Scopus (223) Google Scholar) from the overall rotation correlation time (TR) averaged over 15N nuclei with 1H{15N} NOE > 0.6. NMR spatial structures of the ErbB2tm dimer were calculated using the CYANA program (19Güntert P. Prog. NMR Spectrosc. 2003; 43: 105-125Abstract Full Text Full Text PDF Scopus (224) Google Scholar). Intramonomeric NOE distance restraints (supplemental Fig. S2) were identified with CARA through the analysis of three-dimensional 15N- and 13C-edited NOESY experiments (60- and 80-ms mixing times) (15Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. 2nd Ed. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego, CA2006Google Scholar) performed for 15N- and 15N/13C-labeled samples in H2O and D2O, respectively. The two-dimensional 1H NOESY (80-ms mixing time) spectrum acquired for the unlabeled sample was used as an additional source of structural information concerning aromatic ring protons. Ten (per monomer) unambiguous intermonomeric distance restraints (Figs. 1B and 2B) were derived from three-dimensional 15N-edited NOESY, two-dimensional 15N-13C F1-filtered/F3-edited NOESY, and three-dimensional 13C F1-filtered/F3-edited NOESY spectra (20Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (539) Google Scholar) acquired with a 80-ms mixing time for the 15N-labeled and heterodimer samples in H2O and D2O, respectively. Protein-lipid NOE contacts (Fig. 1C and supplemental Fig. S3) were identified from the three-dimensional 15N-edited NOESY and three-dimensional 13C F1-filtered/F3-edited NOESY spectra acquired with a 80-ms mixing time for the 15N- and 15N/13C-labeled samples in H2O and D2O, respectively. Stereospecific assignments and torsion angle restraints for φ, ψ, and χ1 were obtained by analysis of the local conformation in CYANA using sequential NOE data and the available 3JHNα and 3JNβ coupling constants evaluated quantitatively from three-dimensional 1H-15N HNHA and qualitatively from three-dimensional 1H-15N HNHB experiments (15Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. 2nd Ed. Protein NMR Spectroscopy: Principles and Practice. Academic Press, San Diego, CA2006Google Scholar). Backbone dihedral angle restraints were also estimated based on the assigned chemical shifts using the TALOS program (21Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2740) Google Scholar). The 24 slowly (per monomer) exchanging amide protons (supplemental Fig. S2) were assigned as hydrogen bond donors with related hydrogen acceptor partners on the basis of preliminary structure calculations. Corresponding hydrogen bond restraints were employed in subsequent calculations for d(O,N), d(O,HN), and d(C,HN) distances as described (22Baker E.N. Hubbard R.E. Prog. Biophys. Mol. Biol. 1984; 44: 97-179Crossref PubMed Scopus (1648) Google Scholar). The standard CYANA simulated annealing protocol was applied to 100 random structures, and the resulting 12 structures with the lowest target function were selected. Structure Refinement via Molecular Dynamics Relaxation—Constrained energy relaxation of the NMR structure of the ErbB2tm dimer was performed by molecular dynamics in a hydrated explicit DMPC bilayer using the GROMACS 3.3.3 package (23Lindahl E. Hess B. der Spoel van D. J. Mol. Model. 2001; 7: 306-317Crossref Google Scholar). The protein-lipid system construction and MD protocol are described in the supplemental "Experimental Procedures." The 5-ns MD run for the representative model of the ErbB2tm dimer was carried out with NMR-derived intra- and intermonomeric distance restraints adapted for GROMACS by modification from proton-proton distance restraints to restraints between carbon atoms and polar protons. Furthermore, two additional MD simulations were performed starting from coordinates of the resulting systems. In the first one, the 5-ns continuation MD run without restraints was carried out to check the stability of the dimer. The second simulation (1-ns MD with NMR distance restraints, followed by 5-ns free molecular dynamics) was done for V659E mutant ErbB2tm with a protonated (uncharged) carboxyl group of Glu659 in both dimer subunits. Equilibrium parts of MD trajectories (last 2 ns) were analyzed using original software developed by us and utilities supplied with the GROMACS package. Hydrophobic properties of α-helices were calculated using the molecular hydrophobicity potential approach (see supplemental "Experimental Procedures" for details) (24Efremov R.G. Vergoten G. J. Phys. Chem. 1995; 99: 10658-10666Crossref Scopus (47) Google Scholar). The resulting structures of the ErbB2tm dimer were visualized with MOLMOL (25Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6498) Google Scholar) and PyMOL (35DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar). The contact area between the dimer subunits was calculated using the DSSP program (26Kabsch W. Sander C. Biopolymers. 1985; 22: 2577-2637Crossref Scopus (12414) Google Scholar) as a difference between the accessible surface areas of ErbB2tm residues in the monomer and dimer. Tertiary Fold of the ErbB2tm Dimer in Lipid Bicelles—Recombinant 44-residue ErbB2 fragment 641–684 (named ErbB2tm), including the hydrophobic TM segment flanked by polar N- and C-terminal regions, was solubilized in an aqueous suspension of bicelles consisting of DMPC and DHPC lipids with deuterated hydrophobic tails using DMPC/DHPC and lipid/protein molar ratios of 0.25 and 35, respectively, and then studied with a conventional 15N-13C heteronuclear NMR technique. Circular dichroism spectra (recorded to validate the NMR experimental conditions) proved virtually identical for ErbB2tm incorporated into the DMPC/DHPC bicelles and DMPC unilamellar liposomes (phospholipid bilayer), revealing an α-helical structure of the fragment in the membrane mimetics (supplemental Fig. S1). In addition, the observed protein-lipid NOE contacts with polar lipid heads suggested that the fragment spans the hydrophobic phase of the bicelle (supplemental Fig. S3), therefore mimicking the embedding of the ErbB2 TM domain into a double-layer lipid membrane reasonably well. The 15N{1H} NOE and 15N T1 and T2 values (supplemental Fig. S4) reflecting internal dynamics of the protein backbone were indicative of stable TM segment 651–677, whereas the flanking N- and C-terminal regions had nearly unrestricted mobility. The overall rotation correlation time estimated from the T1/T2 ratio on the TM region is ∼17 ns, which corresponds well with the molecular mass (∼44 kDa) of the ErbB2tm dimer surrounded by ∼70 lipid molecules composing a bicelle with an effective hydrodynamic radius of 24 ± 2 Å measured by dynamic light scattering (supplemental Fig. S1). The unique set of intra- and intermonomeric NOE contacts identified in NMR spectra (Fig. 2B and supplemental Fig. S2) confirmed the helical structure of ErbB2tm in bicelles and directly demonstrated that the TM fragment forms a symmetrical dimer on the NMR time scale with parallel subunits. Therefore, the NMR-derived dihedral angle restraints and both intra- and intermonomeric distance restraints were assigned for each subunit that resulted in a dimer with 2-fold symmetry averaged over the ensemble of calculated structures (Fig. 2A and supplemental Fig. S5). The representative structure of the ErbB2tm dimer was subjected to energy relaxation by molecular dynamics in an explicit hydrated lipid bilayer of DMPC with the experimentally derived distance restraints that allowed adaptation of the structure to the explicit membrane. After constrained energy relaxation, the membrane-spanning α-helices of ErbB2tm cross at an angle θ of -42° with the distance d = 7.7 Å between helix axes, forming a right-handed parallel symmetrical dimer tilted slightly from the membrane normal, whereas the flexible N and C termini of ErbB2 are exposed to water (Fig. 2C). The continuation of molecular dynamics without restraints did not cause considerable change in the dimer structure, indicating its stability in the membrane. A survey of the structural statistics for the final ensemble of the 12 NMR-derived structures of the ErbB2tm dimer and for the representative dimer structure relaxed in the explicit membrane is provided in Table 1.TABLE 1Structural statistics for the ensemble of the 12 best NMR structures of the ErbB2tm dimer and of a representative structure of the ErbB2tm dimer after MD relaxation r.m.s.d., root meant square deviation.NMR distance and dihedral restraints Total unambiguous NOE restraints448Intraresidue186Interresidue264Sequential (|i – j| = 1)142Medium-range (1 < |i – j| ≤ 4)106Long-range (|i – j| > 4)0Intermonomeric NOE20 Hydrogen bond restraints (upper/lower)144/144 Total torsion angle restraints152Backbone φ56Backbone ψ56 Side chain χ140Structure calculation statistics CYANA target function (Å2)0.11 ± 0.01 Restraint violationsDistance (>0.2 Å)0Dihedral (>5°)0 Average pairwise r.m.s.d. (Å)Monomeric stable α-helical region (651–677)2Backbone atoms0.24 ± 0.10All heavy atoms0.78 ± 0.12Dimeric stable α-helical region (651–677)2Backbone atoms0.44 ± 0.16All heavy atoms0.92 ± 0.17 Ramachandran analysisaRamachandran statistics was determined using PROCHECK_NMR (34)% residues in most favored regions80.5% residues in additional allowed regions16.6% residues in generously allowed regions2.7bResidues were from unfolded and flexible regions% residues in disallowed regions0.2bResidues were from unfolded and flexible regions Helix-helix packingContact surface area (Å2)Stable α-helical region (651–677)2390 ± 20Angle θ between TM helix axes–41 ± 2° Distance d (Å) between TM helix axes7.9 ± 0.1Restrained MD relaxation (last 2 ns) Lennard-Jones contact energy (kJ/mol)Stable α-helical region (651–677)2–122 ± 13 Contact surface area (Å2)Stable α-helical region (651–677)2360 ± 30 Angle θ between TM helix axes–42 ± 4° Distance d (Å) between TM helix axes7.7 ± 0.5a Ramachandran statistics was determined using PROCHECK_NMR (34Laskowski R.A. Rullman J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4470) Google Scholar)b Residues were from unfolded and flexible regions Open table in a new tab ErbB2tm Dimerization Interface—The helix-packing surface of the ErbB2tm dimer is rather polar, with some hydrophobic segments on the outer side (Fig. 2, D–F). Being embedded into the DMPC/DHPC bicelles, the ErbB2tm fragment self-associates through a tandem variant of the GG4-like motif Thr652-X3-Ser656-X3-Gly660, located in the N-terminal part of the TM helix (Fig. 2C). The polar contact area of this motif is shielded from lipid tails by the side chains of Leu651, Ile655, Ala657, Val659, and Ile661 (Fig. 2, D–F), whereas the slightly polar concave surface of the C-terminal GG4-like motif Gly668-X3-Gly672 is exposed to the hydrophobic lipid environment (Fig. 2, C–E). The side chains of the opposite Ser656 and Thr652 residues of both dimer subunits are closely packed in the ErbB2tm dimerization interface. Constrained MD relaxation of the ErbB2tm dimer structure revealed two possible configurations of transient intermonomeric hydrogen bonds occurring between the side chains of the Ser656/Ser656* or Thr652/Thr652* pair (Fig. 3, A and B). In addition, the side chain of Ser656 is sometimes involved in intramonomeric hydrogen bonding with the backbone carbonyl group of Thr652. The local conformation of the ErbB2tm dimerization interface is asymmetrical; however, in the course of MD relaxation, hydrogen bond switching occurs. It is accompanied by a change in the hydrogen bond partners through minor rearrangements, so the dimer would be symmetrical on the NMR time scale. Hence, the (Thr652/Ser656)2 hydrophilic motif in the N-terminal part of the dimerization interface forms a node of switching inter- and intramonomeric hydrogen bonds mediating the ErbB2tm helix packing. The C-terminal TM part of the ErbB2tm dimer is stabilized by intermonomeric contacts of the side chains of Leu663, Val664, and Leu667, forming a hydrophobic cluster flanked by the aromatic rings of the opposite Phe671 residues participating in an intermolecular edge-face stacking interaction (Fig. 3C). In addition, during MD relaxation of the ErbB2tm dimer in an explicit DMPC bilayer, multiple transient intermonomeric interactions of the flexible N termini were observed, especially between the strong polar residues Glu645 and Arg647 (Fig. 3D). The existence of such interactions was confirmed experimentally by higher local correlation times compared with the unfolded C termini of ErbB2tm (supplemental Fig. S4D). Thus, the ErbB2 TM helices surrounded by the lipid bicelle interact via the N-terminal tandem GG4-like motif, whereas the C-terminal GG4-like motif is not employed. Apart from these dimerization motifs, presumably associated with the active and inactive states of the receptor (7Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar), the ErbB2 TM domain possesses a leucine zipper heptad motif, Ile-Ile655-X5-Ile-Leu662-X6-Val669 (27Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (122) Google Scholar). This potential dimerization site (28Beevers A. Kukol A. J. Mol. Biol. 2006; 361: 945-953Crossref PubMed Scopus (20) Google Scholar) is located on the lipid-exposed surface of the dimer (Fig. 2, C–E), implying the possibility of the involvement of the ErbB2 TM segment in additional helix-helix interactions with other partners (e.g. in heterodimers or higher order oligomers). ErbB2 TM Domain Conformation and Receptor Activity—The structural properties of the ErbB2tm dimer with a flexible network of hydrogen bonds in the dimerization interface appear to enable the ErbB2 TM domain to undergo conformational switching (4Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar, 11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar, 12Enosh A.R. Fleishman S.J. Ben-Tal N. Halperin D. Bioinformatics (Oxf.). 2006; 23: e212-e218Crossref Scopus (17) Google Scholar) upon receptor activation. The described dimer conformation of the ErbB2 TM domain mediated by the N-terminal GG4-like motif is believed to correspond to the receptor active state (7Mendrola J.M. Berger M.B. King M.C. Lemmon M.A. J. Biol. Chem. 2002; 277: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar). Aberrant activity of ErbB has been implicated in numerous human pathological states, especially in cancer development (2Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). As suggested (11Fleishman S.J. Schlesinger J. Ben-Tal N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15937-15940Crossref PubMed Scopus (233) Google Scholar, 13Smith S.O. Smith C. Shekar S. Peersen O. Ziliox M. Aimoto S. Biochemistry. 2002; 41: 9321-9332Crossref PubMed Scopus (71) Google Scholar, 29Partridge A.W. Therien A.G. Deber C.M. Biopolymers. 2002; 66: 350-358Crossref PubMed Scopus (47) Google Scholar), the I655V single-nucleotide polymorphism associated with increased risk of breast cancer (30Xie D. Shu X.O. Deng Z. Wen W.Q. Creek K.E. Dai Q. Gao Y.T. Jin F. Zheng W. J. Natl. Cancer Inst. 2000; 92: 412-417Crossref PubMed Scopus (157) Google Scholar) and the oncogenic polar mutation V659E observed for the ErbB2 rat homolog (31Bargmann C.I. Hung M.-C. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (812) Google Scholar) could shift the equilibrium between the active and inactive states of the receptor in vivo. According to the obtained structure, both residues participate in dimerization of the ErbB2 TM helices. The I655V variant can excessively stabilize the ErbB2 active dimeric state due to substitution of the bulk side chain of Ile with a smaller one of Val, thus allowing tighter TM helix packing. The V659E mutation can cause the intermonomeric hydrogen bonding of the glutamate side chains with the opposite hydroxyl and backbone carbonyl groups or directly with one another, which would overstabilize dimerization via the N-terminal TM motif, resulting in uncontrolled receptor activation and subsequent cell transformation. Magic angle spinning solid-state NMR studies of the activated pro-oncogenic mutant of the ErbB2 rat homolog revealed that the glutamate carboxyl group is protonated and hydrogen-bonded in the dimerization interface of the TM domain (32Smith S.O. Smith C.S. Bormann B.J. Nat. Struct. Biol. 1996; 3: 252-258Crossref PubMed Scopus (153) Google Scholar). Indeed, MD simulation of the NMR-derived ErbB2tm dimer with a substitution of the Val659 side chains with protonated glutamate side chains revealed that the Glu659 residues can participate in intermonomeric hydrogen bonding without distortion of the helix-packing interface (Fig. 3, E and F), i.e. the ErbB2 TM domain configuration corresponding to the receptor active state is preserved and strengthened. Thus, the obtained ErbB2tm dimer structure explains the biochemical and oncogenic properties of the human ErbB2 receptor and provides a basis to control its kinase activity, which is critical in many disease states. However, additional structural studies of the mutant forms of the ErbB2 TM domain may prove to be essential in the search for related therapeutic agents specifically recognizing the TM helices (33Yin H. Slusky J.S. Berger B.W. Walters R.S. Vilaire G. Litvinov R.I. Lear J.D. Caputo G.A. Bennett J.S. DeGrado W.F. Science. 2007; 315: 1817-1822Crossref PubMed Scopus (244) Google Scholar) and therefore are now in progress. Download .pdf (.52 MB) Help with pdf files