TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry.

SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.

Integrating Hydrogen Deuterium Exchange-Mass Spectrometry with Molecular Simulations Enables Quantification of the Conformational Populations of the Sugar Transporter XylE.

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilizing them for in vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange-mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our strategy on wild-type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modeling to capture, accurately quantify, and subsequently visualize co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors.

Structural dynamics in the evolution of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein-Lipid Assemblies.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.

A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments.

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87-118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Size-Exclusion Chromatography for Analysis of Complex Protein Samples.

The growing use of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for studying membrane proteins, large protein assemblies, and highly disulfide-bonded species is often challenged by the presence in the sample of large amounts of lipids, protein ligands, and/or highly ionizable reducing agents. Here, we describe how a short size-exclusion chromatography (SEC) column can be integrated with a conventional temperature-controlled HDX-MS setup to achieve fast and online removal of unwanted species from the HDX sample prior to chromatographic separation and MS analysis. Dual-mode valves permit labeled proteins eluting after SEC to be directed to the proteolytic and chromatographic columns, while unwanted sample components are led to waste. The SEC-coupled HDX-MS method allows analyses to be completed with lower or similar back-exchange compared to conventional experiments. We demonstrate the suitability of the method for the analysis of challenging protein samples, achieving efficient online removal of lipid components from protein-lipid systems, depletion of an antibody from an antigen during epitope mapping, and elimination of MS interfering compounds such as tris(2-carboxyethyl)phosphine (TCEP) during HDX-MS analysis of a disulfide-bonded protein. The implementation of the short SEC column to the conventional HDX-MS setup is straightforward and could be of significant general utility during the HDX-MS analysis of complex protein states.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

Optimization of a Top-Down Proteomics Platform for Closely Related Pathogenic Bacterial Discrimination.

The current technique used for microbial identification in hospitals is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, it suffers from important limitations, in particular, for closely related species or when the database used for the identification lacks the appropriate reference. In this work, we set up a liquid chromatography (LC)-MS/MS top-down proteomics platform, which aims at discriminating closely related pathogenic bacteria through the identification of specific proteoforms. Using Escherichia coli as a model, all steps of the workflow were optimized: protein extraction, on-line LC separation, MS method, and data analysis. Using optimized parameters, about 220 proteins, corresponding to more than 500 proteoforms, could be identified in a single run. We then used this platform for the discrimination of enterobacterial pathogens undistinguishable by MALDI-TOF, although leading to very different clinical outcomes. For each pathogen, we identified specific proteoforms that could potentially be used as biomarkers. We also improved the characterization of poorly described bacterial strains. Our results highlight the advantage of addressing proteoforms rather than peptides for accurate bacterial characterization and qualify top-down proteomics as a promising tool in clinical microbiology. Data are available via ProteomeXchange with the identifier PXD019247.

Transfer of Nicotine, Cotinine and Caffeine Into Breast Milk in a Smoker Mother Consuming Caffeinated Drinks.

Although the habits of cigarette smoking and associated coffee drinking are generally ceased during pregnancy, they are often reinitiated after delivery when the breastfeeding period starts. This is a case report of a 32-year-old lactating smoker mother who consumed caffeinated drinks and who agreed to donate breast milk after smoking one cigarette (containing 0.6 mg of nicotine) and drinking one cup of espresso (containing 80 mg of caffeine) for an investigation of the excretion of nicotine, its major metabolite cotinine and caffeine into the breast milk and subsequent transfer to the infant. Nicotine and its metabolite cotinine peaked in the breast milk at 0.5 h after the cigarette smoking, and caffeine peaked 2 h after drinking coffee. Moreover, the nicotine disappeared from the milk by 3 h, the caffeine required 24 h and the cotinine required 72 h. The relative infant doses of caffeine, nicotine and cotinine were found to be 8.9, 12.8 and 77.6%, respectively. In the light of these results obtained after the mother smoked only one cigarette and consumed one cup of espresso, if a lactating mother cannot refrain from smoking cigarettes, she should extend the time between the last smoked cigarette and breastfeeding to at least 3 h when the nicotine has been completely eliminated from the milk. Similarly, nursing mothers should also drink coffee sparingly and immediately after nursing and avoid coffee or caffeinated beverages for at least 4 h prior to breastfeeding to minimize the infant's exposure to caffeine.

Fetal exposure to ethanol: relationship between ethyl glucuronide in maternal hair during pregnancy and ethyl glucuronide in neonatal meconium.

BACKGROUND: In recent years, fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG) in meconium emerged as reliable, direct biological markers for establishing gestational ethanol exposure. We investigated whether EtG in maternal hair measured during the three trimesters of pregnancy correlated with EtG and FAEEs in neonatal meconium. METHODS: In a prospective sample of 80 mother-infant dyads from Barcelona (Spain), we measured EtG and FAEE in maternal hair segments and meconium samples using a validated UHPLC-MS/MS method. RESULTS: Fifty-eight (72.5%) women had EtG concentrations in the hair shafts >7 pg/mg in one or more pregnancy trimesters, and EtG and FAEEs in meconium samples were documented in 50 and 24 of their neonates, respectively. The best significant correlations (p<0.0001) were found between EtG concentration in the proximal 0-3 and 3-6 hair shaft segments corresponding to the last two pregnancy trimesters and EtG in neonatal meconium (ρ=0.609 and ρ=0.577, respectively). Using the combination of EtG in meconium ≥30 ng/g and a median of EtG >11 pg/mg in maternal hair during the second and third trimesters of pregnancy, prenatal ethanol exposure could be predicted with a sensitivity of 85.7% and specificity of 73.7%. CONCLUSIONS: This study provides evidence of proven fetal exposure to ethanol during the second and third trimesters of pregnancy by linking detection of ethanol biomarkers (EtG) in maternal hair segments and EtG in neonatal meconium.

Drugs of abuse in maternal hair and paired neonatal meconium: an objective assessment of foetal exposure to gestational consumption.

In a prospective sample of 80 mother-infant dyads, we investigated whether drugs of abuse in maternal hair measured during the pregnancy trimesters were also present in neonatal meconium. Principal drugs of abuse were analyzed in the three consecutive maternal hair segments and meconium samples by ultra-performance liquid chromatography tandem mass spectrometry assay. Of the 80 mothers, 32 (40%) presented one or more hair shafts with at least one of the analyzed drugs of abuse and/or its metabolites. The drug of abuse with a higher prevalence in our study population was methamphetamine: 19 mothers had methamphetamine in one or more hair segments (59.4%). The second most detected drug of abuse was cocaine; nine mothers presented cocaine in one or more hair segments (28.1%). Nineteen pregnant women consumed at least one drug of abuse during the first trimester, ten continued consuming drugs of abuse during the second trimester; and nine consumed until the end of pregnancy. Five of the nine newborns from mothers who consumed drugs during the whole pregnancy showed drugs of abuse in meconium samples. Newborns from the 23 remaining mothers with one or two hair shafts positive to drugs of abuse did not present drugs in their meconium. Indeed from these results, it seems that discontinuous and/or sporadic consumption during pregnancy could produce a negligible transplacental passage and hence negative results in meconium. Furthermore, the role of placenta in the metabolism and excretion of drugs of abuse is still to be precisely investigated. Copyright © 2015 John Wiley & Sons, Ltd.