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.

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.

Lipid binding attenuates channel closure of the outer membrane protein OmpF.

Strong interactions between lipids and proteins occur primarily through association of charged headgroups and amino acid side chains, rendering the protonation status of both partners important. Here we use native mass spectrometry to explore lipid binding as a function of charge of the outer membrane porin F (OmpF). We find that binding of anionic phosphatidylglycerol (POPG) or zwitterionic phosphatidylcholine (POPC) to OmpF is sensitive to electrospray polarity while the effects of charge are less pronounced for other proteins in outer or mitochondrial membranes: the ferripyoverdine receptor (FpvA) or the voltage-dependent anion channel (VDAC). Only marginal charge-induced differences were observed for inner membrane proteins: the ammonia channel (AmtB) or the mechanosensitive channel. To understand these different sensitivities, we performed an extensive bioinformatics analysis of membrane protein structures and found that OmpF, and to a lesser extent FpvA and VDAC, have atypically high local densities of basic and acidic residues in their lipid headgroup-binding regions. Coarse-grained molecular dynamics simulations, in mixed lipid bilayers, further implicate changes in charge by demonstrating preferential binding of anionic POPG over zwitterionic POPC to protonated OmpF, an effect not observed to the same extent for AmtB. Moreover, electrophysiology and mass-spectrometry-based ligand-binding experiments, at low pH, show that POPG can maintain OmpF channels in open conformations for extended time periods. Since the outer membrane is composed almost entirely of anionic lipopolysaccharide, with similar headgroup properties to POPG, such anionic lipid binding could prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways.

Peptide-Based Approach to Inhibition of the Multidrug Resistance Efflux Pump AcrB.

Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix-helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrB-mediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Förster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein-protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies.

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

Membrane proteins bind lipids selectively to modulate their structure and function.

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea. We reveal that ATP-free HerA and HerA-DNA complexes predominantly exist in solution as a heptamer and act as a DNA loading intermediate. The binding of either NurA or ATP stabilizes the hexameric HerA, indicating that HerA-NurA is activated by substrates and complex assembly. To examine the role of ATP in DNA translocation and processing, we investigated how nucleotides interact with the HerA-NurA. We show that while the hexameric HerA binds six nucleotides in an 'all-or-none' fashion, HerA-NurA harbors a highly coordinated pairwise binding mechanism and enables the translocation and processing of double-stranded DNA. Using molecular dynamics simulations, we reveal novel inter-residue interactions between the external ATP and the internal DNA binding sites. Overall, here we propose a stepwise assembly mechanism detailing the synergistic activation of HerA-NurA by ATP, which allows efficient processing of double-stranded DNA.

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments.

Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted systems which lack the native lipid membrane and cellular environments. However, there has been progress in the use and adaptations of these methods for probing membrane proteins within increasingly more native contexts. In this Concept article the use and utility of structural mass spectrometry techniques for studying membrane proteins within native environments are highlighted.

Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions.

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene-maleic acid lipid particle (SMALP) technology can be coupled with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.

Elucidation of Drug Metabolite Structural Isomers Using Molecular Modeling Coupled with Ion Mobility Mass Spectrometry.

Ion mobility-mass spectrometry (IM-MS) in combination with molecular modeling offers the potential for small molecule structural isomer identification by measurement of their gas phase collision cross sections (CCSs). Successful application of this approach to drug metabolite identification would facilitate resource reduction, including animal usage, and may benefit other areas of pharmaceutical structural characterization including impurity profiling and degradation chemistry. However, the conformational behavior of drug molecules and their metabolites in the gas phase is poorly understood. Here the gas phase conformational space of drug and drug-like molecules has been investigated as well as the influence of protonation and adduct formation on the conformations of drug metabolite structural isomers. The use of CCSs, measured from IM-MS and molecular modeling information, for the structural identification of drug metabolites has also been critically assessed. Detection of structural isomers of drug metabolites using IM-MS is demonstrated and, in addition, a molecular modeling approach has been developed offering rapid conformational searching and energy assessment of candidate structures which agree with experimental CCSs. Here it is illustrated that isomers must possess markedly dissimilar CCS values for structural differentiation, the existence and extent of CCS differences being ionization state and molecule dependent. The results present that IM-MS and molecular modeling can inform on the identity of drug metabolites and highlight the limitations of this approach in differentiating structural isomers.

Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.

Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase.

Despite the growing importance of the mass spectrometry of membrane proteins, it is not known how their transfer from solution into vacuum affects their stability and structure. To address this we have carried out a systematic investigation of ten membrane proteins solubilized in different detergents and used mass spectrometry to gain physicochemical insight into the mechanism of their ionization and desolvation. We show that the chemical properties of the detergents mediate the charge state, both during ionization and detergent removal. Using ion mobility mass spectrometry, we monitor the conformations of membrane proteins and show how the surface charge density dictates the stability of folded states. We conclude that the gas-phase stability of membrane proteins is increased when a greater proportion of their surface is lipophilic and is consequently protected by the physical presence of the micelle.

Mg2+-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg2+, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg2+ and pH in modulating the dynamics of the periplasmic adaptor protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug efflux pump from Escherichia coli. In the absence of Mg2+, AcrA becomes increasingly plastic within acidic conditions, but when Mg2+ is bound this is ameliorated, resulting in domain specific organisation in neutral to weakly acidic regimes. We establish a unique histidine residue directs these structural dynamics and is essential for sustaining pump efflux activity across acidic, neutral, and alkaline conditions. Overall, we propose Mg2+ conserves the structural mobility of AcrA to ensure optimal AcrAB-TolC function within rapid changing environments commonly faced by the periplasm during bacterial infection and colonization. This work highlights that Mg2+ is an important mechanistic component in this pump class and possibly across other periplasmic lipoproteins.

Structural mass spectrometry approaches to understand multidrug efflux systems.

Multidrug efflux pumps are ubiquitous across both eukaryotes and prokaryotes, and have major implications in antimicrobial and multidrug resistance. They reside within cellular membranes and have proven difficult to study owing to their hydrophobic character and relationship with their compositionally complex lipid environment. Advances in structural mass spectrometry (MS) techniques have made it possible to study these systems to elucidate critical information on their structure-function relationships. For example, MS techniques can report on protein structural dynamics, stoichiometry, connectivity, solvent accessibility, and binding interactions with ligands, lipids, and other proteins. This information proving powerful when used in conjunction with complementary structural biology methods and molecular dynamics (MD) simulations. In the present review, aimed at those not experts in MS techniques, we report on the current uses of MS in studying multidrug efflux systems, practical considerations to consider, and the future direction of the field. In the first section, we highlight the importance of studying multidrug efflux proteins, and introduce a range of different MS techniques and explain what information they yield. In the second section, we review recent studies that have utilised MS techniques to study and characterise a range of different multidrug efflux systems.

Conformational restriction shapes the inhibition of a multidrug efflux adaptor protein.

Membrane efflux pumps play a major role in bacterial multidrug resistance. The tripartite multidrug efflux pump system from Escherichia coli, AcrAB-TolC, is a target for inhibition to lessen resistance development and restore antibiotic efficacy, with homologs in other ESKAPE pathogens. Here, we rationalize a mechanism of inhibition against the periplasmic adaptor protein, AcrA, using a combination of hydrogen/deuterium exchange mass spectrometry, cellular efflux assays, and molecular dynamics simulations. We define the structural dynamics of AcrA and find that an inhibitor can inflict long-range stabilisation across all four of its domains, whereas an interacting efflux substrate has minimal effect. Our results support a model where an inhibitor forms a molecular wedge within a cleft between the lipoyl and αß barrel domains of AcrA, diminishing its conformational transmission of drug-evoked signals from AcrB to TolC. This work provides molecular insights into multidrug adaptor protein function which could be valuable for developing antimicrobial therapeutics.

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.

Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes.

In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies. Alteration of the lipid bilayer composition improves the efficiency of the folding system. The approach also enables membrane transport proteins to be made and inserted into artificial cell platforms such as droplet interface bilayers. Importantly, this gives a new facet to the droplet networks by enabling specific transport of molecules across the synthetic bilayer against a concentration gradient. This method also includes a protocol to pause and restart translation of membrane proteins at specified positions during their co-translational folding. This stop-start strategy provides an avenue to investigate whether the proteins fold in sequence order, or if the correct fold of N-terminal regions is reliant on the synthesis of downstream residues.

Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB.

Resistance-nodulation-division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

Direct protein-lipid interactions shape the conformational landscape of secondary transporters.

Secondary transporters undergo structural rearrangements to catalyze substrate translocation across the cell membrane - yet how such conformational changes happen within a lipid environment remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with molecular dynamics (MD) simulations to understand how lipids regulate the conformational dynamics of secondary transporters at the molecular level. Using the homologous transporters XylE, LacY and GlpT from Escherichia coli as model systems, we discover that conserved networks of charged residues act as molecular switches that drive the conformational transition between different states. We reveal that these molecular switches are regulated by interactions with surrounding phospholipids and show that phosphatidylethanolamine interferes with the formation of the conserved networks and favors an inward-facing state. Overall, this work provides insights into the importance of lipids in shaping the conformational landscape of an important class of transporters.