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 ␣-helical bundle through the N-terminal tandem GG4-like motif Thr 652 -X 3 -Ser 656 -X 3 -Gly 660 , 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 (1). 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 (2). 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 (3, 4). Biochemical and genetic studies have revealed that the single-helix transmembrane (TM) 3 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 (5, 6). Mutational analysis assumed that the dimerization involves consensus small-X 3 -small (so-called GG4-like) motifs, formed by residues with small side chains allowing tight helix packing (7-9). 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 (4, 10, 11). 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 ␣-helical TM segment, employing different GG4-like motifs for dimerization (7,(11)(12)(13). 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.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 actio...

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.

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ␣-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and threefinger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 M, ws-LYNX1 competed with 125 I-␣-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ␣7 nAChRs to 1 M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ␣4␤2 and ␣3␤2 nAChRs. Increasing ws-LYNX1 concentration to 10 M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.Endogenous "prototoxins" like LYNX1, LYNX2, SLURP-1, and SLURP-2, belonging to the Ly6 protein family, modulate nicotinic acetylcholine receptors (nAChRs) 3 (1-8). In the central nervous system, LYNX1 and LYNX2 regulate nAChR activity, preventing excessive excitation (3, 4). Gene deletion of LYNX1 or LYNX2 indicates that these modulators are critical for nAChR function in the brain. LYNX1 knock-out mice demonstrated enhanced performance in specific tests of learning ability and memory, whereas loss of LYNX2 results in increased anxiety-related behaviors (3, 4). Prototoxins have also been shown to affect cell growth in lung carcinoma (9), are involved in skin diseases (6, 7), and are related to prostate stem cell antigen (10).LYNX1 and LYNX2 are tethered to the membrane by a GPI anchor, which considerably complicates in vitro studies. LYNX1 is co-localized in the brain with ␣4␤2 and ␣7 nAChRs (1-3), and its modulatory activity on ␣4␤2 nAChR was shown in experiments on Xenopus oocytes (1, 3). It was reported that soluble form of LYNX1 (not containing a GPI anchor) potentiates ␣4␤2 receptor (1), but the concentration at which it acts remains unknown. A secreted water-soluble protein SLURP-1 expressed in palmoplantar skin acts on ␣7 nAChR and regulates keratinocyte proliferation (5).It was predicted that the prototoxins should have a spatial structure similar to that of snake venom ␣-neurotoxins, effective competitive inhibitors of nAChR (1). ␣-Neurotoxins are characterized by a three-finger fold formed by three adjacent loops arising from a small globular hydrophobic core, crosslinked by four conserved disulfide bonds (11-13). Nicotinic acetylcholine receptors are ta...

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Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.