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.

An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time tau(R) from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional tau(R) determination method from T(1)/T(2) ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 +/- 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T(1)/T(2) ratio.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

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One-bond residual dipolar couplings (RDCs) measured for the amide groups of proteins partially aligned in a magnetic field provide valuable information regarding the relative orientation of protein units. In order for RDCs obtained for individual proteins to be useful in the structure determination of heterodimer complexes, they should be measured for exactly the same alignment of the complex. Here, an isotopically discriminated IDIS-RDC-TROSY NMR experiment is proposed, which enables the measurement of HN RDCs for two proteins simultaneously and independently, but in the same sample, while they are part of the same complex. The signals for both proteins, one of which should be labeled with (15)N and the other with (15)N and (13)C, are observed in different subspectra, thus reducing spectral overlap. The approach uniquely ensures that RDCs measured for both proteins relate to exactly the same alignment tensor, allowing accurate measurement of the relative angle between the two proteins. The method is also applicable for complexes containing three or more protein components. The experiment can speed up and lead to automation of protein-protein docking on the basis of angular restraints.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

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