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...

Eph receptors are found in a wide variety of cells in developing and mature tissues and represent the largest family of receptor tyrosine kinases, regulating cell shape, movements, and attachment. The receptor tyrosine kinases conduct biochemical signals across plasma membrane via lateral dimerization in which their transmembrane domains play an important role. Structural-dynamic properties of the homodimeric transmembrane domain of the EphA1 receptor were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics in explicit lipid bilayer. EphA1 transmembrane segments associate in a right-handed parallel ␣-helical bundle, region (544 -569) 2 , through the N-terminal glycine zipper motif A 550 X 3 G 554 X 3 G 558 . Under acidic conditions, the N terminus of the transmembrane helix is stabilized by an N-capping box formed by the uncharged carboxyl group of Glu 547 , whereas its deprotonation results in a rearrangement of hydrogen bonds, fractional unfolding of the helix, and a realignment of the helix-helix packing with appearance of additional minor dimer conformation utilizing seemingly the C-terminal GG4-like dimerization motif A 560 X 3 G 564 . This can be interpreted as the ability of the EphA1 receptor to adjust its response to ligand binding according to extracellular pH. The dependence of the pK a value of Glu 547 and the dimer conformational equilibrium on the lipid head charge suggests that both local environment and membrane surface potential can modulate dimerization and activation of the receptor. This makes the EphA1 receptor unique among the Eph family, implying its possible physiological role as an "extracellular pH sensor," and can have relevant physiological implications.Erythropoietin-producing hepatocellular (Eph) 3 receptor and corresponding membrane-bound Eph receptor-interacting proteins (ephrins) transduce signal in a cell-cell contact-dependent fashion, thereby coordinating growth, differentiation, and patterning of almost every organ and tissue during vertebrate and invertebrate embryogenesis (1, 2). In adult organism, Eph-ephrin interactions can also trigger a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion, especially for neuronal and endothelial cells, whereas deregulated reemergence of Eph function appears to contribute to mechanism of tissue injury and of tumor invasion and metastasis. Intriguingly the Eph-ephrin interactions may have a role in synaptic plasticity, learning, memory formation, and mental disease (3, 4). The Eph receptors represent the largest family of receptor tyrosine kinases and are divided into subclasses A and B based on the sequence homology of their extracellular parts, the structure, and the binding affinity (5). Ephrin-A ligands share a membrane-tethered glycosylphosphatidylinositol anchor, whereas ephrin-B ligands have a transmembrane domain and a short cytoplasmic tail. The Eph receptors and ephrins are not only numerous, but their relationship is also complex (6). Receptor-liga...

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.

Supplementary data are available at Bioinformatics online.

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.Mitochondria hold a crucial role in programmed cell death required to control cell development and to maintain homeostasis in multicellular organisms (1). Mitochondria-mediated cell death is both promoted and suppressed by apoptotic proteins of the Bcl-2 family, most of which contain a C-terminal hydrophobic domain essential for membrane targeting (2). A major function of Bcl-2 family proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homo-and hetero-oligomers inside the membrane that determine cell fate (3-5). The pro-apoptotic protein BNip3 (Bcl-2 Nineteen-kDa interacting protein 3) with a single Bcl-2 homology 3 (BH3) domain is one of the most intensively studied members of the family (6). BNip3 and its homologues belong to an independent monophyletic branch with individual evolutionary history (2) and are essentially different from other BH3-only proteins such as Bid/Bik not only in that they do not require BH3 domain for their function but also because they directly cause changes of mitochondrial potential (7). BNip3-induced cell death is independent of caspases and cytochrome c release; it is believed to represent a novel form of programmed cell death, resembling necrosis rather than classical apoptosis (8).For all cells, loss of nutrient supply represents a potent signal for programmed death. BNip3 plays an important role in hypoxic cell death of normal and malignant cells (9). Hypoxia induces expression and accumulation of cytoplasmic or loosely membrane-bound BNip3; however, in order to activate cell death pathway acidosis is required (10). Transition from respiratory to glycolytic metabolism with increased glucose consumption, lactic acid production, and decrease of cytosolic pH causes redistribution of BNip3 to the outer mitochondrial membrane and integration of homodimeric BNip3 into it, triggering a cell death cascade, which ultimately leads to opening of the mitochondrial permeability tra...

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Surface-enhanced Raman (SER) spectra of water-soluble proteins (lysozyme and bovine serum albumin), dipeptides and amino acids were analysed. Chemisorption is a necessary condition for the appearance of SER spectra on silver electrodes and hydrosols for these compounds. The Raman cross-section enhancement per molecule may reach a factor of 10s-106, depending closely on the frequency of the vibration band considered. The mechanism of enhancement has a short-range character and is attributed to the rc-electron complexes of the aromatic amino acids sidechains and u-complexes of the molecular group containing unsbared electron pairs with the metal. The effect of induced optical activity in the visible region for aromatic amino acids adsorbed by silver hydrosols has been elucidated. (iii) What are the lowest concentrations for the detection of SER soectra of biomolecules in different INTRODUCTIONIt has recently been d e m~n s t r a t e d '~~ that very large (in some instances up to lo9) enhancements of the Raman cross-section for molecules in the close vicinity of a metal surface result from the superposition of two main mechanisms, described as electromagnetic and molecular (or 'chemical'). Enhancement of the local electromagnetic field near 'rough' metal surface induces the electromagnetic mechanism, whereas the 'molecular' mechanism is connected with the appearance of new excited states for the molecule-metal complexes in the process of chemisorption.Surface-enhanced Raman (SER) and surfaceenhanced resonance Raman (SERR) spectroscopy are based on these mechanisms and have been widely used in investigations of biological molecule^.^-^ Three important questions relating to the applicability of SERS and SERRS to sophisticated problems in molecular biology, bioorganic and physical chemistry are the following :(i) What are the molecular mechanisms of interaction of the biomolecules with the metal surface under the experimental conditions typical of SERS appearance and is it possible to make measurements preserving the native conformation of the molecule? (ii) What is the real dependence of the Raman crosssection enhancement on the distance between the metal and the molecule? Is the mechanism of enhancement short-range or long-range and will it be possible to detect all normal vibrations of macromolecules or only vibrations of groups which directly contact the surface? experimental systems (electrodes, hydrosols, surfaces with the regular roughnesses)? Is it possible to detect high-quality SER spectra of subpicogram amounts of different classes of biomolecules for successful competition with the traditional techniques in biotechnology and genetic engineering? This paper deals with applications of SERS to the study of water-soluble proteins adsorbed on silver electrodes. A comparison of the spectra of some amino acids and model compounds was made and a new effect, induced by adsorption optical activity in the visible region, was revealed. In subsequent papers in this series the possibility of the selective application of ...

SUMMARY The insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH>7.9. The alkali-sensing property of IRR is conserved in frog, mouse and human. IRR activation is specific, dose-dependent, quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess.