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. 2010 Mar 3;98(5):881-9.
doi: 10.1016/j.bpj.2009.11.008.

Left-handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity Among Receptor Tyrosine Kinases

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Free PMC article

Left-handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity Among Receptor Tyrosine Kinases

Eduard V Bocharov et al. Biophys J. .
Free PMC article

Abstract

The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.

Figures

Figure 1
Figure 1
NMR spectrum of the EphA2tm dimer embedded into lipid bicelles. The 1H-15N HSQC spectrum of 1 mM 15N-labeled EphA2tm in DMPC/DHPC (1/4) bicelles at 40°C and pH 5.0. The 1H-15N backbone resonance assignments are shown. The amide crosspeaks of solvent-exposed N-terminal residues have a minor component (marked by asterisk) due to slow cis/trans transitions of Ser528-Pro529 peptide bond in the flexible N-terminal part of EphA2tm.
Figure 2
Figure 2
NMR data for the EphA2tm dimer embedded into lipid bicelles. (A) Intramonomeric sequential and medium range NOE connectivity observed in 3D 15N- and 13C-edited NOESY-HSQC spectra (80 ms mixing time) are shown by horizontal lines. The line thickness for the NOE connectivity is inversely proportional to the squared upper distance bound. The NOE information on some regions was restricted due to crosspeak broadening and overlapping. (B) The residues (solid stars) showing NOE contacts between their side chains and polar lipid heads. (C) Strong (solid circle) and weak (open circle) crosspeaks detected on the water frequency for the EphA2tm amide groups in the 15N-edited NOESY- and TOCSY-HSQC spectra. The crosspeaks result from direct NOE, exchange-relayed NOE or chemical exchange (21) of the amide protons with water and anyway indicate water accessibility of the EphA2tm residue. (D) Slowly hydrogen-deuterium exchanging amide groups in the EphA2tm dimer according to estimated half-exchange times (40°C, pH 5.0): 0.5 < t1/2 < 4 h (open box); t1/2 > 4 h (solid box). The data for the Phe556, Phe557, and Ile558 residues subjected to HN resonance broadening were not obtained. (E) 1Hα secondary chemical shifts Δδ for the EphA2tm residues given by the difference between actual chemical shift and typical random-coil chemical shift for a given residue. Pronounced negative 1Hα secondary chemical shifts indicate a helical structure of the protein (21). (F) Steady-state 15N{1H} NOE for the backbone 15N nuclei of the EphA2tm dimer. Uncertainties are shown by bars. (G) Effective rotation correlation times τR for the EphA2tm amide groups calculated from the ratio of 15N longitudinal T1 and transverse T2 relaxation times (Fig. S2).
Figure 3
Figure 3
Spatial structure of the EphA2tm dimer. (A) Ensemble of 15 NMR-derived structures of the EphA2tm dimer after superposition of the backbone atoms of residues (535–559)2 of both dimer subunits. Side chain (in red and blue for different monomers) and backbone (in black) heavy atom bonds of residues (532–562)2 are shown. The EphA2tm dimer structure is presented in detail on Fig. S4. (B) Ribbon diagrams of the EphA2tm dimer relaxed in the explicit DPPC bilayer. Yellow balls show phosphorus atoms of lipid heads. Spatial locations of the two dimerization motifs, heptad repeat motif L535X3G539X2A542X3V546X2L549 used in the EphA2tm dimerization and the potential N-terminal tandem GG4-like (glycine zipper) motif A536X3G540X3G544, are marked by dashed ovals. Phe-ring patch composed of (FF557)2 in the C-terminal part of the EphA2tm dimer interface is also highlighted. (C) Local conformational exchange near the Phe-ring patch (also see Fig. S6). Side chains of Phe556, Phe557, Phe556′, and Phe557′ of the EphA2tm dimer subunits from the three representative NMR structures are colored in black, red, green, and blue, respectively. Side chains of histidine and arginine residues are colored in purple. Amide groups of Gly553, Val554, Phe556, and Ile558 having 1H{15N} NOE > 0.8 are highlighted in cyan. Amide groups of Phe556, Phe557, and Ile558 subjected to resonance broadening are presented by thick cylinders. (D) Hydrophobic and hydrophilic (polar) surfaces of TM helices of the EphA2tm dimer colored in yellow and green according to the MHP. Four water molecules penetrating into the weekly polar cavity located in the dimeric interface are shown. (E) Hydrophobicity map for the EphA2tm helix surface with contour isolines encircling hydrophobic regions with high values of MHP. Details about map construction are presented in the Supporting Material. The EphA2tm helix packing interface is indicated by red-point area. The potential dimerization glycine zipper motif is marked by dashed green oval. (F) TM helix packing contact surface per EphA2tm residue. The heptad repeat and glycine zipper motifs are highlighted in red and green, respectively. (G) Schematic illustration of membrane thickness preference of the EphA2 TM domain on dimerization via heptad repeat or glycine zipper motifs resulting in the left- or right-handed conformations, respectively, with different overall dimer shape.

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