Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1–Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1–Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1–Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

The Tibellus oblongus spider is an active predator that does not spin webs and remains poorly investigated in terms of venom composition. Here, we present a new toxin, named Tbo-IT2, predicted by cDNA analysis of venom glands transcriptome. The presence of Tbo-IT2 in the venom was confirmed by proteomic analyses using the LC-MS and MS/MS techniques. The distinctive features of Tbo-IT2 are the low similarity of primary structure with known animal toxins and the unusual motif of 10 cysteine residues distribution. Recombinant Tbo-IT2 (rTbo-IT2), produced in E. coli using the thioredoxin fusion protein strategy, was structurally and functionally studied. rTbo-IT2 showed insecticidal activity on larvae of the housefly Musca domestica (LD100 200 μg/g) and no activity on the panel of expressed neuronal receptors and ion channels. The spatial structure of the peptide was determined in a water solution by NMR spectroscopy. The Tbo-IT2 structure is a new example of evolutionary adaptation of a well-known inhibitor cystine knot (ICK) fold to 5 disulfide bonds configuration, which determines additional conformational stability and gives opportunities for insectotoxicity and probably some other interesting features.