Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogenMycobacterium tuberculosis(Mtb). While this lipid promotes the entry ofMtbinto macrophages, which occurs via phagocytosis, its molecular mechanism of action is unknown. Here, we combined biophysical, cell biology, and modeling approaches to reveal the molecular mechanism of DIM action on macrophage membranes leading to the first step ofMtbinfection. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry showed that DIM molecules are transferred from theMtbenvelope to macrophage membranes during infection. Multiscale molecular modeling and31P-NMR experiments revealed that DIM adopts a conical shape in membranes and aggregates in the stalks formed between 2 opposing lipid bilayers. Infection of macrophages pretreated with lipids of various shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken together, these results reveal how the molecular shape of a mycobacterial lipid can modulate the biological response of macrophages.

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.

24Phthiocerol dimycocerosate (DIM) is a major virulence factor of the pathogen 25 Mycobacterium tuberculosis (Mtb). While this lipid promotes the entry of Mtb into 26 macrophages, which occurs via phagocytosis, its molecular mechanism of action is 27 unknown. Here, we combined biophysical, cell biology, and modelling approaches to 28 reveal the molecular mechanism of DIM action on macrophage membranes leading to 29 the first step of Mtb infection. MALDI-TOF mass spectrometry showed that DIM 30 molecules are transferred from the Mtb envelope to macrophage membranes during 31 infection. Multi-scale molecular modeling and 31 P-NMR experiments revealed that DIM 32 adopts a conical shape in membranes and aggregate in the stalks formed between 33 two opposing lipid bilayers. Infection of macrophages pre-treated with lipids of various 34shapes uncovered a general role for conical lipids in promoting phagocytosis. Taken 35 together, these results reveal how the molecular shape of a mycobacterial lipid can 36 modulate the biological function of macrophages. 37 38 we developed a multidisciplinary approach combining multiscale Molecular Dynamics 73 (MD) simulations, solid-state NMR and cell biology experiments. This revealed how 74 the molecular shape of DIM can affect macrophage membranes to promote 75 phagocytosis. 76 77 78 RESULTS 79 80 DIM are transferred to host cell membranes during macrophage infection 81First, we used MALDI-TOF mass spectrometry to assess whether DIM added 82 to host cells is incorporated into their membranes. Human macrophage (THP-1) cells 83 Figure 1: DIM are transferred from the bacterial envelope to macrophage membranes. (a) Structure of the DIM family of lipids, where m denotes the range of carbon atoms on the phthiocerol moiety, and n and p on the mycocerosate moieties. (b) MALDI-TOF mass spectra of purified DIM and of the membrane fraction of macrophages treated with DIM. (c) MALDI-TOF mass spectra of WT Mtb (HR37v) and of the membrane fraction of macrophages infected by H37Rv or by the H37RvDlppX mutant. M: low intensity peak corresponding to the detection of the matrix molecule in the DIM region of interest. The star symbol highlights the mass of the DIM molecule chosen for the modelling, with m=18, n=17, and p=4.

PEGylation is a promising approach to address the central challenge of applying biologics, i.e., lack of protein stability in the demanding environment of the human body. Wider application is hindered by lack of atomic level understanding of protein-PEG interactions, preventing design of conjugates with predicted properties. We deployed an integrative structural and biophysical approach to address this critical challenge with the PEGylated carbohydrate recognition domain of human galectin-3 (Gal3C), a lectin essential for cell adhesion and potential biologic. PEGylation dramatically increased Gal3C thermal stability, forming a stable intermediate and redirecting its unfolding pathway. Structural details revealed by NMR pointed to a potential role of PEG localization facilitated by charged residues. Replacing these residues subtly altered the protein-PEG interface and thermal unfolding behavior, providing insight into rationally designing conjugates while preserving PEGylation benefits.

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) structureactivity 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 modelling 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.

Protein−polymer conjugates are widely used in many clinical and industrial applications, but lack of experimental data relating protein−polymer interactions to improved protein stability prevents their rational design. Advances in synthetic chemistry have expanded the palette of polymer designs, including development of nonlinear architectures, novel monomer chemical scaffolds, and control of hydrophobicity, but more experimental data are needed to transform advances in chemistry into next generation conjugates. Using an integrative biophysical approach, we investigated the molecular basis for polymer-based thermal stabilization of a human galectin protein, Gal3C, conjugated with polymers of linear and nonlinear architectures, different degrees of polymerization, and varying hydrophobicities. Independently varying the degree of polymerization and polymer architecture enabled delineation of specific polymer properties contributing to improved protein stability. Insights from NMR spectroscopy of the polymer-conjugated Gal3C backbone revealed patterns of protein−polymer interactions shared between linear and nonlinear polymer architectures for thermally stabilized conjugates. Despite large differences in polymer chemical scaffolds, protein−polymer interactions resulting in thermal stabilization appear conserved. We observed a clear relation between polymer length and protein− polymer thermal stability shared among chemically different polymers. Our data indicate a wide range of polymers may be useful for engineering conjugate properties and provide conjugate design criteria.

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