A dimerization hierarchy in the transmembrane domains of the HER receptor family.

Bitopic membrane proteins offer an opportunity for studying transmembrane domain interactions without the structural complexity inherent to multitopic integral membrane proteins. To date, only homomeric associations have been extensively studied quantitatively. Here we propose to assess the thermodynamics of heteromeric associations, which opens the way to investigating specificity and selectivity. A very interesting system of biological relevance with single transmembrane domains possibly involved in interactions with different partners is the EGFR receptor family. The four members, all tyrosine kinase receptors, are involved in an interaction network that potentially leads to a complete set of homo- and heterodimers, ideally suited to such a study. Furthermore, the transmembrane domains of these receptors have been previously implicated in their function in the past by mutations in the transmembrane domain leading to constitutive activation. We demonstrate, using a fluorescence-based measurement of interaction energies, a hierarchy of transmembrane domain interactions ranging from a noninteractive pair to strong dimerization. We propose a structural model based on the crystal structure of the EGFR dimer, to show how the dimeric structure favors these interactions. The correlation we observe between transmembrane domain and whole receptor interaction hierarchies opens a new perspective, suggesting a role for transmembrane receptor domains in the modulation of receptor signaling.

Hydrogen bonding in a model bacteriochlorophyll-binding site drives assembly of light harvesting complex.

In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position -4 relative to the liganding histidine of the alpha-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix. A hydrogen bond between the hydroxy group of the serine and the 13(1) keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.

Spectroscopic characterisation of a tetrameric subunit form of the core antenna protein from Rhodospirillum rubrum.

The core light-harvesting complex (LH1) of Rhodospirillum rubrum is constituted of multiple heterodimeric subunits, each containing two transmembrane polypeptides, alpha and beta. The detergent octylglucoside induces the stepwise dissociation of LH1 into B820 (an alphabeta dimer) and B777 (monomeric polypeptides), both of which still retain their bound bacteriochlorophyll molecules. We have investigated the absorption properties of B820 as a function of temperature, whereby a spectral population called 'B851' has been characterised. We show evidence that it is a dimer of the B820 complex. This may represent an intermediate oligomeric form in the process of the LH1 ring formation, as its existence was predicted from global analysis of the absorption spectra of the LH1/B820 equilibrium [Pandit et al. (2001) Biochemistry 40, 12913-12924]. Stabilisation of this dissociated form of LH1 may help in understanding both the electronic properties and the association process of these integral membrane proteins.