Large-Scale Conformational Changes of FhaC Provide Insights Into the Two-Partner Secretion Mechanism.

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane ß barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein's surface loops. Specifically, our data indicate that an amphipathic POTRA2 ß hairpin can insert into the ß barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

Molecular insights into mechanisms of GPCR hijacking by Staphylococcus aureus.

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus bicomponent pore-forming leukotoxins to promote bacterial growth and immune evasion. Here, we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, using cell-based assays and native mass spectrometry, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding with the extracellular domain of ACKR1. Unexpectedly, hydrogen/deuterium exchange mass spectrometry analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation and/or changes in the architecture of ACKR1-Gαi1 protein complexes observed in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR.

Intact monoclonal antibodies separation and analysis by sheathless capillary electrophoresis-mass spectrometry.

Capillary electrophoresis-mass spectrometry coupling is a growing technique in biopharmaceutics characterization. Assessment of monoclonal antibodies is well known at middle-up and bottom-up levels to obtain information about the sequence, post-translational modifications and degradation products. Intact protein analysis is an actual challenge to be closer to the real protein structure. At this level, actual techniques are time consuming or cumbersome processes. In this work, a 20 minutes separation method has been developed to optimize characterization of intact monoclonal antibodies. Thus, separation has been done on a positively charged coated capillary with optimized volatile background electrolyte and sample buffer. Three world-wide health authorities approved monoclonal antibodies have been used to set up a rapid and ease of use method. Intact trastuzumab, rituximab and palivizumab isoforms have been partially separated with this method in less than 20 minutes under denaturing conditions. For each monoclonal antibody, 2X-glycosylated and 1X-glycosylated structures have been identified and separated. Concerning basic and acidic variants, potential aspartic acid isomerization modification and asparagine deamidation have been observed. Accurate mass determination for high-mass molecular species remains a challenge, but the progress in intact monoclonal antibodies separation appears very promising for biopharmaceutics characterization.