Database of glutamate gated chloride (GluCl) subunits across 125 nematode species: patterns of gene accretion and sequence diversification

Glutamate Gated Chloride (GluCl) channels belong to the Cys-loop receptor superfamily. GluCl channels are activated by glutamate (Glu) and form substrates for the anti-parasitic drugs from the avermectin family. GluCl channels are pentameric, and each subunit contains an N-terminal extracellular domain that binds Glu and four helical transmembrane domains (TMs), which contain binding sites for avermectin drugs. In order to provide more insight into phylum-wide patterns of GluCl subunit gene expansion and sequence diversity across nematodes, we have developed a database of predicted GluCl subunit genes from 125 nematode species. Our analysis into this dataset described assorted patterns of species-specific GluCl gene counts across different nematodes as well as sequence diversity in key residues thought to be involved in avermectin binding.

Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and to validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAM tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface.

Comparison of carbohydrate ABC importers from Mycobacterium tuberculosis

Background Mycobacterium tuberculosis, the etiological agent of tuberculosis, has at least four ATP-Binding Cassette (ABC) transporters dedicated to carbohydrate uptake: LpqY/SugABC, UspABC, Rv2038c-41c, and UgpAEBC. LpqY/SugABC transporter is essential for M. tuberculosis survival in vivo and potentially involved in the recycling of cell wall components. The three-dimensional structures of substrate-binding proteins (SBPs) LpqY, UspC, and UgpB were described, however, questions about how these proteins interact with the cognate transporter are still being explored. Components of these transporters, such as SBPs, show high immunogenicity and could be used for the development of diagnostic and therapeutic tools. In this work, we used a phylogenetic and structural bioinformatics approach to compare the four systems, in an attempt to predict functionally important regions. Results Through the analysis of the putative orthologs of the carbohydrate ABC importers in species of Mycobacterium genus it was shown that Rv2038c-41c and UgpAEBC systems are restricted to pathogenic species. We showed that the components of the four ABC importers are phylogenetically separated into four groups defined by structural differences in regions that modulate the functional activity or the interaction with domain partners. The regulatory region in nucleotide-binding domains, the periplasmic interface in transmembrane domains and the ligand-binding pocket of the substrate-binding proteins define their substrates and segregation in different branches. The interface between transmembrane domains and nucleotide-binding domains show conservation of residues and charge. Conclusions The presence of four ABC transporters in M. tuberculosis dedicated to uptake and transport of different carbohydrate sources, and the exclusivity of at least two of them being present only in pathogenic species of Mycobacterium genus, highlights their relevance in virulence and pathogenesis. The significant differences in the SBPs, not present in eukaryotes, and in the regulatory region of NBDs can be explored for the development of inhibitory drugs targeting the bacillus. The possible promiscuity of NBDs also contributes to a less specific and more comprehensive control approach.

Structural insights into the activation of human calcium-sensing receptor

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of 7-transmembrane domains, and inter- and intrasubunit conformational changes of 7-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

Solid-State NMR Studies of the Succinate-Acetate Permease from Citrobacter Koseri in Liposomes and Native Nanodiscs

The succinate-acetate permease (SatP) is an anion channel with six transmembrane domains. It forms different oligomers, especially hexamers in the detergent as well as in the membrane. Solid-state NMR studies of SatP were carried out successfully on SatP complexes by reconstructing the protein into liposomes or retaining the protein in the native membrane of E. Coli., where it was expressed. The comparison of 13C-13C 2D correlation spectra between the two samples showed great similarity, opening the possibility to further study the acetate transport mechanism of SatP in its native membrane environment. Solid-state NMR studies also revealed small chemical shift differences of SatP in the two different membrane systems, indicating the importance of the lipid environment in determining the membrane protein structures and dynamics. Combining different 2D SSNMR spectra, chemical shift assignments were made on some sites, consistent with the helical structures in the transmembrane domains. In the end, we pointed out the limitation in the sensitivity for membrane proteins with such a size, and also indicated possible ways to overcome it.

Efficient integration of transmembrane domains depends on the folding properties of the upstream sequences

The topology of most membrane proteins is defined by the successive integration of α-helical transmembrane domains at the Sec61 translocon. The translocon provides a pore for the transfer of polypeptide segments across the membrane while giving them lateral access to the lipid. For each polypeptide segment of ∼20 residues, the combined hydrophobicities of its constituent amino acids were previously shown to define the extent of membrane integration. Here, we discovered that different sequences preceding a potential transmembrane domain substantially affect its hydrophobicity requirement for integration. Rapidly folding domains, sequences that are intrinsically disordered or very short or capable of binding chaperones with high affinity, allow for efficient transmembrane integration with low-hydrophobicity thresholds for both orientations in the membrane. In contrast, long protein fragments, folding-deficient mutant domains, and artificial sequences not binding chaperones interfered with membrane integration, requiring higher hydrophobicity. We propose that the latter sequences, as they compact on their hydrophobic residues, partially folded but unable to reach a native state, expose hydrophobic surfaces that compete with the translocon for the emerging transmembrane segment, reducing integration efficiency. The results suggest that rapid folding or strong chaperone binding is required for efficient transmembrane integration.

Smuggle tau through a secret(ory) pathway

Secretion of misfolded tau, a microtubule-binding protein enriched in nerve cells, is linked to the progression of tau pathology. However, the molecular mechanisms underlying tau secretion are poorly understood. Recent work by Lee et al. [Biochemical J. (2021) 478: 1471–1484] demonstrated that the transmembrane domains of syntaxin6 and syntaxin8 could be exploited for tau release, setting a stage for testing a novel hypothesis that has profound implications in tauopathies (e.g. Alzheimer's disease, FTDP-17, and CBD/PSP) and other related neurodegenerative diseases. The present commentary highlights the importance and limitations of the study, and discusses opportunities and directions for future investigations.

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.