ABSTRACT During infection, host immune cells deploy a variety of strategies to neutralize invading pathogens, including the manipulation of metal availability, a process traditionally understood as nutritional immunity. While depriving microbes of essential metals, such as iron and manganese, inhibits their growth, host cells also engage in metal intoxication, actively overloading phagosomes with toxic levels of transition metals, such as copper and zinc. To survive these dual pressures, Mycobacterium tuberculosis , the etiological agent of tuberculosis, has evolved specialized metal resistance mechanisms. This review explores how M. tuberculosis counters host-imposed metal stress through an arsenal of P-type ATPases, particularly the diverse P 1B subfamily of transition metal exporters. We detail the structural features, metal specificities, and regulatory mechanisms of M. tuberculosis ’ 12 P-type ATPases, focusing on three key systems, CtpC, CtpG, and CtpV, and their cognate scaffold proteins PacL1, PacL2, and PacL3. These PacL-Ctp pairs form dynamic membrane assemblies termed effluxosomes, which mediate resistance to transition metals such as zinc and cadmium. The review also highlights several distinctive features of M. tuberculosis P 1B -ATPases relative to canonical transporters such as CopA and ZntA, suggesting unique adaptations to the intracellular environment. Finally, we discuss the challenges of functionally and structurally characterizing these systems and propose future directions to elucidate effluxosome assembly and function. Together, these insights reveal how M. tuberculosis leverages metal export as a critical survival strategy and suggest novel therapeutic opportunities targeting metal detoxification pathways.

The dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed in myeloid cells. Although patient and experimental data implicates DCIR in diverse diseases, including chronic inflammation, autoimmunity, allergy, infection, and cancer, the physiological ligand of DCIR remains elusive, leaving both its precise role in immunoregulation and its potential for therapeutic targeting unclear. Here, we identify the low-density lipoprotein receptor-related protein 1 (LRP1), a ubiquitously expressed, highly glycosylated membrane receptor, as a conserved endogenous ligand for human DCIR and murine DCIR1. DCIR specifically interacts with LRP1 through galactose-terminated biantennary complex-type N-glycans. By combining X-ray crystallography, small angle X-ray scattering, and site-directed mutagenesis, we define the structural organization of DCIR as a dimeric receptor and the basis of DCIR-glycan recognition, revealing key contact residues and an atypical glycan-binding mode. We further demonstrate that DCIR is activated through cis-interactions with its ligand upon co-engagement of ITAM-containing receptors, such as Dectin-1 or FcγRs, and modulates FcγR-mediated phagocytosis. Overall, our study addresses a long-standing gap by identifying the physiological ligand of DCIR, uncovering a novel glyco-immune recognition axis, and opening avenues for targeted intervention in DCIR-associated diseases.

SUMMARY The dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor expressed by myeloid cells that plays a key immunoregulatory role in a wide range of diseases, from inflammation to cancer. However, the ligand(s) of DCIR remain(s) unknown, hampering our understanding of the exact function of this immune receptor. Here, we found that both human DCIR and mouse DCIR1 bind specifically to the low-density lipoprotein receptor-related protein 1 (LRP1), a heavily glycosylated receptor mediating the clearance of various molecules from the extracellular matrix and apoptotic cells. This interaction is mediated by galactose-terminated biantennary complex-type N-glycans, including those carrying the immunogenic α-Gal epitope. Our study provides a deeper understanding of the role of DCIR in immune regulation and its potential impact on a range of immune disorders, highlighting its specificity in ligand recognition which is crucial for developing therapeutic strategies.

Macrophage inducible Ca2+-dependent lectin (Mincle) receptor recognizes Mycobacterium tuberculosis glycolipids to trigger an immune response. This host membrane receptor is thus a key player in the modulation of the immune response to infection by M. tuberculosis and has emerged as a promising target for the development of new vaccines against tuberculosis. The recent development of the Martini 3 force field for coarse-grained (CG) molecular modeling allows the study of interactions of soluble proteins with small ligands which was not typically modeled well with the previous Martini 2 model. Here, we present a refined approach detailing a protocol for modeling interactions between a glycolipid and its receptor at a CG level using the Martini 3 force field. Using this approach, we studied Mincle and identified critical parameters governing ligand recognition, such as loop flexibility and the regulation of hydrophobic groove formation by calcium ions. In addition, we assessed ligand affinity using free energy perturbation calculations. Our results offer mechanistic insight into the interactions between Mincle and glycolipids, providing a basis for the rational design of molecules targeting this type of membrane receptors.

The human pathogen Mycobacterium tuberculosis requires a P1B-ATPase metal exporter, CtpC (Rv3270), for resistance to zinc poisoning. Here, we show that zinc resistance also depends on a chaperone-like protein, PacL1 (Rv3269). PacL1 contains a transmembrane domain, a cytoplasmic region with glutamine/alanine repeats and a C-terminal metal-binding motif (MBM). PacL1 binds Zn2+, but the MBM is required only at high zinc concentrations. PacL1 co-localizes with CtpC in dynamic foci in the mycobacterial plasma membrane, and the two proteins form high molecular weight complexes. Foci formation does not require flotillin nor the PacL1 MBM. However, deletion of the PacL1 Glu/Ala repeats leads to loss of CtpC and sensitivity to zinc. Genes pacL1 and ctpC appear to be in the same operon, and homologous gene pairs are found in the genomes of other bacteria. Furthermore, PacL1 colocalizes and functions redundantly with other PacL orthologs in M. tuberculosis. Overall, our results indicate that PacL proteins may act as scaffolds that assemble P-ATPase-containing metal efflux platforms mediating bacterial resistance to metal poisoning.

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.

OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of β-barrel proteins for several decades. In Escherichia coli , OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal β-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results suggest a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.

Dynorphin is a neuropeptide involved in pain, addiction and mood regulation. It exerts its activity by binding to the kappa opioid receptor (KOP) which belongs to the large family of G protein-coupled receptors. The dynorphin peptide was discovered in 1975, while its receptor was cloned in 1993. This review will describe: (a) the activities and physiological functions of dynorphin and its receptor, (b) early structure-activity relationship studies performed before cloning of the receptor (mostly pharmacological and biophysical studies of peptide analogues), (c) structure-activity relationship studies performed after cloning of the receptor via receptor mutagenesis and the development of recombinant receptor expression systems, (d) structural biology of the opiate receptors culminating in X-ray structures of the four opioid receptors in their inactive state and structures of MOP and KOP receptors in their active state. X-ray and EM structures are combined with NMR data, which gives complementary insight into receptor and peptide dynamics. Molecular modeling greatly benefited from the availability of atomic resolution 3D structures of receptor-ligand complexes and an example of the strategy used to model a dynorphin-KOP receptor complex using NMR data will be described. These achievements have led to a better understanding of the complex dynamics of KOP receptor activation and to the development of new ligands and drugs.

The development of drug delivery and imaging tools is a major challenge in human health, in particular in cancer pathologies. This work describes the optimization of a protein nanocontainer, belonging to the lectin protein family, for its use in epithelial cancer diagnosis and treatment. Indeed, it specifically targets a glycosidic marker, the T antigen, which is known to be characteristic of epithelial cancers. Its quaternary structure reveals a large hydrated inner cavity able to transport small therapeutic molecules. Optimization of the nanocontainer by site directed mutagenesis allowed controlling loading and release of confined drugs. Doxorubicin confinement was followed, both theoretically and experimentally, and provided a proof of concept for the use of this nanocontainer as a vectorization system. In OVCAR-3 cells, a human ovarian adenocarcinoma cell line that expresses the T antigen, the drug was observed to be delivered inside late endosomes/lysosomes. These results show that this new type of vectorization and imaging device opens new exciting perspectives in nano-theranostic approaches.

Ghrelin plays a central role in controlling major biological processes. As for other G protein-coupled receptor (GPCR) peptide agonists, the structure and dynamics of ghrelin bound to its receptor remain obscure. Using a combination of solution-state NMR and molecular modeling, we demonstrate that binding to the growth hormone secretagogue receptor is accompanied by a conformational change in ghrelin that structures its central region, involving the formation of a well-defined hydrophobic core. By comparing its acylated and nonacylated forms, we conclude that the ghrelin octanoyl chain is essential to form the hydrophobic core and promote access of ghrelin to the receptor ligand-binding pocket. The combination of coarse-grained molecular dynamics studies and NMR should prove useful in improving our mechanistic understanding of the complex conformational space explored by a natural peptide agonist when binding to its GPCR. Such information should also facilitate the design of new ghrelin receptor-selective drugs.

G-protein coupled receptors (GPCR) play an essential role in human physiology. They are prominent pharmacological targets and the understanding of molecular mechanisms underlying their activity is fundamental for the design of new drugs. Since a decade, a number of structures have been elucidated, but sparse in complex with peptide agonists. NMR spectroscopy allows determining conformations of peptides bound to their receptors. Here, we report our work on Ghrelin, a lipopeptide hormone involved in phenomena such as appetite, growth hormone secretion and reward-seeking behaviours. Our study led to the determination of Ghrelin's structure and dynamics when bound to the growth hormone secretagogue receptor (GHSR). The perdeuterated receptor reconstituted into lipid nanodiscs after expression in E. coli allowed studying Ghrelin in its bound state by liquid state NMR. We performed transferred 1H NOE experiments to determine the hormone's conformation and we measured 15N transverse relaxation to decipher the conformational flexibility along its sequence. Consistent with pharmacological data, Ghrelin amino-terminal part folds with its acyl chain to form a hydrophobic core essential for GHSR binding and activation. In contrast, the carboxy-terminal part remains flexible and may participate in the binding with the receptor through electrostatic interactions. Furthermore, we are combining NMR data with molecular dynamics simulations to construct a model of the Ghrelin-GHSR complex. This approach provides the first active structure of Ghrelin and a novel insight into the molecular mechanisms responsible for its activity with respect to the activation of GHSR. Considering the pharmacological relevance of Ghrelin and its receptor, our results may lead to the design of new drugs with applications in the treatment of obesity, diabetes and addiction.

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The role of membrane proteins in cellular mechanism strongly depends on their dynamics, and solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is a unique method to exhaustively characterize motions of proteins in a lipid environment. Herein, we make use of advances in 1H-detected MAS NMR to describe the dynamics of the membrane domain of the Outer membrane protein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar-coupling as well as 15N R1 and R1ρ relaxation rates at fast (60 kHz) MAS and high magnetic field (1 GHz), we were able to describe the motions of the residues of the β-barrel as a collective rocking of low amplitude and of hundreds of nanoseconds time scale. Residual local motions at the edges of the strands, underscored by enhanced 15N R1ρ relaxation rates, report on the mobility of the connected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, reconstituted in liposomes or in detergent micelles, revealed in all cases the existence of a unique trypsin cleavage site within the membrane domain (out of 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, showing that it is highly accessible to protein-protein interactions. KpOmpA is involved in cell-cell recognition, for adhesion and immune response mechanisms. The L3 region may therefore play a key role in pathogenicity.