Advances in the mass spectrometry of membrane proteins: from individual proteins to intact complexes.

Rapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.

The Adsorption of P2X2 Receptors Interacting with IgG Antibodies Revealed by Combined AFM Imaging and Mechanical Simulation.

The adsorption of proteins onto surfaces significantly impacts biomaterials, medical devices, and biological processes. This study aims to provide insights into the irreversible adsorption process of multiprotein complexes, particularly focusing on the interaction between anti-His6 IgG antibodies and the His6-tagged P2X2 receptor. Traditional approaches to understanding protein adsorption have centered around kinetic and thermodynamic models, often examining individual proteins and surface coverage, typically through Molecular Dynamics (MD) simulations. In this research, we introduce a computational approach employing Autodesk Maya 3D software for the investigation of multiprotein complexes' adsorption behavior. Utilizing Atomic Force Microscopy (AFM) imaging and Maya 3D-based mechanical simulations, our study yields real-time structural and kinetic observations. Our combined experimental and computational findings reveal that the P2X2 receptor-IgG antibody complex likely undergoes absorption in an 'extended' configuration. Whereas the P2X2 receptor is less adsorbed once is complexed to the IgG antibody compared to its individual state, the opposite is observed for the antibody. This insight enhances our understanding of the role of protein-protein interactions in the process of protein adsorption.

Ultrastructural characterisation of young and aged dental enamel by atomic force microscopy.

Recent advances in atomic force microscopy (AFM) have allowed the characterisation of dental-associated biomaterials and biological surfaces with high resolution. In this context, the topography of dental enamel - the hardest mineralised tissue in the body - has been explored with AFM-based approaches at the microscale. With age, teeth are known to suffer changes that can impact their structural stability and function; however, changes in enamel structure because of ageing have not yet been explored with nanoscale resolution. Therefore, the aim of this exploratory work was to optimise an approach to characterise the ultrastructure of dental enamel and determine potential differences in topography, hydroxyapatite (HA) crystal size, and surface roughness at the nanoscale associated to ageing. For this, a total of six teeth were collected from human donors from which enamel specimens were prepared. By employing intermittent contact (AC mode) imaging, HA crystals were characterised in both transversal and longitudinal orientation (respect to surface plane) with high resolution in environmental conditions. The external enamel surface displayed the presence of a pellicle-like coating on its surface that was not observable on cleaned specimens. Acid-etching exposed crystals that were imaged and morphologically characterised in high resolution at the nanoscale in both the external and internal regions of enamel in older and younger specimens. Our results demonstrated important individual variations in HA crystal width and roughness parameters across the analysed specimens; however, an increase in surface roughness and decrease in HA width was observed for the pooled older external enamel group compared to younger specimens. Overall, high-resolution AFM was an effective approach for the qualitative and quantitative characterisation of human dental enamel ultrastructure. Future work should focus on exploring the ageing of dental enamel with increased sample sizes to compensate for individual differences as well as other potential confounding factors such as behavioural habits and mechanical forces.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

KEY POINTS: Extracellular ATP, in association with [Ca2+ ]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. ABSTRACT: Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+ ]i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+ ]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50 U ml-1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1-100 µm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1 mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+ ]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors adopt different subunit arrangements.

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.

Alternative molecular mechanisms for force transmission at adherens junctions via ß-catenin-vinculin interaction.

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, ß-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that ß-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and ß-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving ß-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

Crosstalking interactions between P2X4 and 5-HT3A receptors.

Ionotropic receptors are ligand-gated ion channels triggering fast neurotransmitter responses. Among them, P2X and 5-HT3 receptors have been shown to physically interact each other and functionally inducing cross inhibitory responses. Nevertheless, despite the importance of P2X4 and 5-HT3A receptors that mediate for example neuropathic pain and psychosis respectively, complementary evidence has recently started to move forward in the understanding of this interaction. In this review, we discuss current evidence supporting the mechanism of crosstalking between both receptors, from the structural to the transduction pathway level. We expect this work may guide the design of further experiments to obtain a comprehensive view for the neuropharmacological role of these interacting receptors. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Fractional Factorial Design to Evaluate the Synthesis and Electrochemical Transfer Parameters of h-BN Coatings.

In this study, we present a fractional factorial design approach for exploring the effects and interactions of key synthesis and electrochemical transfer parameters on the roughness and wettability of hexagonal boron nitride (h-BN) coatings, due to their essential role in biofilm formation. The studied parameters for the synthesis process include precursor mass, growth time, and substrate conditioning, whereas for the transfer process, applied voltage and aqueous medium concentration were studied. Through this polynomial model, we confirmed the strong influence of precursor mass and medium concentration parameters on h-BN surface roughness and its resulting antibiofilm properties.

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions.

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

How far are we from predicting multi-drug interactions during treatment for COVID-19 infection?

Seriously ill patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hospitalized in intensive care units (ICUs) are commonly given a combination of drugs, a process known as multi-drug treatment. After extracting data on drug-drug interactions with clinical relevance from available online platforms, we hypothesize that an overall interaction map can be generated for all drugs administered. Furthermore, by combining this approach with simulations of cellular biochemical pathways, we may be able to explain the general clinical outcome. Finally, we postulate that by applying this strategy retrospectively to a cohort of patients hospitalized in ICU, a prediction of the timing of developing acute kidney injury (AKI) could be made. Whether or not this approach can be extended to other diseases is uncertain. Still, we believe it represents a valuable pharmacological insight to help improve clinical outcomes for severely ill patients.

Drug-dependent inhibition of nucleotide hydrolysis in the heterodimeric ABC multidrug transporter PatAB from Streptococcus pneumoniae.

The bacterial heterodimeric ATP-binding cassette (ABC) multidrug exporter PatAB has a critical role in conferring antibiotic resistance in multidrug-resistant infections by Streptococcus pneumoniae. As with other heterodimeric ABC exporters, PatAB contains two transmembrane domains that form a drug translocation pathway for efflux and two nucleotide-binding domains that bind ATP, one of which is hydrolysed during transport. The structural and functional elements in heterodimeric ABC multidrug exporters that determine interactions with drugs and couple drug binding to nucleotide hydrolysis are not fully understood. Here, we used mass spectrometry techniques to determine the subunit stoichiometry in PatAB in our lactococcal expression system and investigate locations of drug binding using the fluorescent drug-mimetic azido-ethidium. Surprisingly, our analyses of azido-ethidium-labelled PatAB peptides point to ethidium binding in the PatA nucleotide-binding domain, with the azido moiety crosslinked to residue Q521 in the H-like loop of the degenerate nucleotide-binding site. Investigation into this compound and residue's role in nucleotide hydrolysis pointed to a reduction in the activity for a Q521A mutant and ethidium-dependent inhibition in both mutant and wild type. Most transported drugs did not stimulate or inhibit nucleotide hydrolysis of PatAB in detergent solution or lipidic nanodiscs. However, further examples for ethidium-like inhibition were found with propidium, novobiocin and coumermycin A1, which all inhibit nucleotide hydrolysis by a non-competitive mechanism. These data cast light on potential mechanisms by which drugs can regulate nucleotide hydrolysis by PatAB, which might involve a novel drug binding site near the nucleotide-binding domains.

Antibacterial Effect of Honey-Derived Exosomes Containing Antimicrobial Peptides Against Oral Streptococci.

PURPOSE: Recently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci. METHODS: A. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure. RESULTS: Molecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans. CONCLUSION: Honey is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilm-modulating strategy in dental caries.

Initial adhesion suppression of biofilm-forming and copper-tolerant bacterium Variovorax sp. on laser microtextured copper surfaces.

The growth of detrimental biofilms on metal surfaces affects their structural performance and lifespan. Microtopographic texturization has emerged as an approach to suppress biofilm growth by preventing the initial stages of bacterial adhesion. This work studies the effects of linear pattern copper texturization on the initial adhesion steps of the biofilm-forming and copper-resistant bacterium Variovorax sp. Linear patterns with 4.7, 6.8, 14, and 18 µm periodicity were produced by direct laser interference patterning (DLIP) on copper coupons. Surface features were characterized by microscopic and spectroscopic techniques, and bacterial adhesion behavior was characterized by epifluorescence microscopy and functionalization of atomic force microscopy tips. We found a periodicity of 4.7 µm as the most efficient pattern to suppress Variovorax sp. initial adhesion by 31.1 % with respect to the nontextured surface. Preferential settlement in hummocks over hollows was observed for patterns with 14 and 18 µm periodicity, with adhesion events showing higher frequency in these topographies than patterns with periodicities of 4.7 and 6.8 µm. Our results highlight an understanding of the initial bacteria-copper adhesion and settlement behavior, thus contributing to the potential development of innocuous strategies for controlling biofilm growth on copper-based materials.

Separating and visualising protein assemblies by means of preparative mass spectrometry and microscopy.

Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to 'soft-land' these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained.

Ion mobility mass spectrometry of two tetrameric membrane protein complexes reveals compact structures and differences in stability and packing.

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.

The subunit arrangement and assembly of ionotropic receptors.

Ionotropic receptors mediate rapid communication between neurons. These receptors are oligomers and are usually assembled from multiple subunit types. Receptors built from different subunit combinations have distinct functional properties, such as single-channel conductances, rates of desensitization and sensitivities to activators and inactivators; they can also have different intracellular locations. Methods are now available for determining not only the subunit stoichiometry but also the subunit arrangement within ionotropic receptors. This information will inform experiments designed to understand the molecular basis of receptor assembly and function. It will also permit the modelling of potential ligand-binding sites at the interfaces between the subunits and should lead to a more rational approach to drug development.

Activation of P2X4 receptors induces an increase in the area of the extracellular region and a decrease in receptor mobility.

The P2X4 receptor (P2X4R) is an ATP-gated cation channel. Here, we used fast-scan atomic force microscopy (AFM) to visualize changes in the structure and mobility of individual P2X4Rs in response to activation. P2X4Rs were purified from detergent extracts of transfected cells and integrated into lipid bilayers. Activation resulted in a rapid (2 s) and substantial (10-20 nm2 ) increase in the cross-sectional area of the extracellular region of the receptor and a corresponding decrease in receptor mobility. Both effects were blocked by the P2X4R antagonist 5-BDBD. Addition of cholesterol to the bilayer reduced receptor mobility, although the ATP-induced reduction in mobility was still observed. We suggest that the observed responses to activation may have functional consequences for purinergic signalling.