ИСТИНА

Toll-like receptors (TLRs) are one of the key players in both the innate and adaptive immune systems. These proteins recognize conserved products unique to microbial metabolism or viral nucleic acids and induce activation of inflammatory and antimicrobial innate immune responses. TLRs are type I transmembrane proteins. Each of them consists of the extracellular domain (ECD) containing leucine-rich repeats that mediate the recognition of pathogen-associated molecular patterns (PAMPs); single transmembrane α-helix domain; and intracellular Toll–interleukin 1 receptor domain (TIR) required for downstream signal transduction. It was shown that TLRs form homo- or heterodimeric signaling complexes interacting with the single PAMP molecule. The medical and biological significance of TLR signaling is obvious, since the disregulation of the TLR system may cause various autoimmune diseases and septic shock, and some therapeutic strategies targeting TLRs have already emerged. There is a lot of data available on the structural and biological aspects of the TLR signaling. Structural analysis of several TLRs have elucidated the mechanisms of PAMP recognition by TLR. But mainly these data concern to the roles of the ECD and TIR domains. There is only one computer model of the dimeric full-length TLR3 receptor in the active state. But the role of the transmembrane domain (TMD) in TLR signaling is still elusive, while its significance for the TLR activation was demonstrated in recent studies. Isolated TMDs of all TLR receptors were shown to homodimerize in bacterial membranes, with TMDs of TLR2,3,8,9 having the highest propensity to perform homotypic interactions. Taking into account all aforesaid, it is obvious that the structural investigations of TLR TMD dimers or oligomers are necessary, because these domains can serve as targets for emerging therapies. In this study the efficient cell-free expression system of TLR3 and TLR4 TMDs and purification protocols are described. The ability of these TMDs to form dimers and trimers in detergent micelles was shown by solution NMR spectroscopy. Spatial structures and free energy magnitudes were determined for both TMDs in dimeric and trimeric states. Possible surfaces of TMDs that may be used for the helix–helix interaction by the full-length receptors were presented. The work is supported by Russian Science Foundation (project #14-14-00573).