Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions.

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF.

Optimization Workflow for the Analysis of Cross-Linked Peptides Using a Quadrupole Time-of-Flight Mass Spectrometer.

Cross-linking mass spectrometry is an emerging structural biology technique. Almost exclusively, the analyzer of choice for such an experiment has been the Orbitrap. We present an optimized protocol for the use of a Synapt G2-Si for the analysis of cross-linked peptides. We first tested six different energy ramps and analyzed the fragmentation behavior of cross-linked peptides identified by xQuest. By combining the most successful energy ramps, cross-link yield can be increased by up to 40%. When compared to previously published Orbitrap data, the Synapt G2-Si also offers improved fragmentation of the ß peptide. In order to improve cross-link quality control we have also developed ValidateXL, a programmatic solution that works with existing cross-linking software to improve cross-link quality control.

The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7.

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.

Structure of the stationary phase survival protein YuiC from B.subtilis.

BACKGROUND: Stationary phase survival proteins (Sps) were found in Firmicutes as having analogous domain compositions, and in some cases genome context, as the resuscitation promoting factors of Actinobacteria, but with a different putative peptidoglycan cleaving domain. RESULTS: The first structure of a Firmicute Sps protein YuiC from B. subtilis, is found to be a stripped down version of the cell-wall peptidoglycan hydrolase MltA. The YuiC structures are of a domain swapped dimer, although some monomer is also found in solution. The protein crystallised in the presence of pentasaccharide shows a 1,6-anhydrodisaccharide sugar product, indicating that YuiC cleaves the sugar backbone to form an anhydro product at least on lengthy incubation during crystallisation. CONCLUSIONS: The structural simplification of MltA in Sps proteins is analogous to that of the resuscitation promoting factor domains of Actinobacteria, which are stripped down versions of lysozyme and soluble lytic transglycosylase proteins.

The C-terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell.

The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N-terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N-terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N-terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite.

Towards absolute quantification of allergenic proteins in food--lysozyme in wine as a model system for metrologically traceable mass spectrometric methods and certified reference materials.

Current routine food allergen quantification methods, which are based on immunochemistry, offer high sensitivity but can suffer from issues of specificity and significant variability of results. MS approaches have been developed, but currently lack metrological traceability. A feasibility study on the application of metrologically traceable MS-based reference procedures was undertaken. A proof of concept involving proteolytic digestion and isotope dilution MS for quantification of protein allergens in a food matrix was undertaken using lysozyme in wine as a model system. A concentration of lysozyme in wine of 0.95 +/- 0.03 microg/g was calculated based on the concentrations of two peptides, confirming that this type of analysis is viable at allergenically meaningful concentrations. The challenges associated with this promising method were explored; these included peptide stability, chemical modification, enzymatic digestion, and sample cleanup. The method is suitable for the production of allergen in food certified reference materials, which together with the achieved understanding of the effects of sample preparation and of the matrix on the final results, will assist in addressing the bias of the techniques routinely used and improve measurement confidence. Confirmation of the feasibility of MS methods for absolute quantification of an allergenic protein in a food matrix with results traceable to the International System of Units is a step towards meaningful comparison of results for allergen proteins among laboratories. This approach will also underpin risk assessment and risk management of allergens in the food industry, and regulatory compliance of the use of thresholds or action levels when adopted.

A multiplex protein panel assay for severity prediction and outcome prognosis in patients with COVID-19: An observational multi-cohort study.

Background: Global healthcare systems continue to be challenged by the COVID-19 pandemic, and there is a need for clinical assays that can help optimise resource allocation, support treatment decisions, and accelerate the development and evaluation of new therapies. Methods: We developed a multiplexed proteomics assay for determining disease severity and prognosis in COVID-19. The assay quantifies up to 50 peptides, derived from 30 known and newly introduced COVID-19-related protein markers, in a single measurement using routine-lab compatible analytical flow rate liquid chromatography and multiple reaction monitoring (LC-MRM). We conducted two observational studies in patients with COVID-19 hospitalised at Charité - Universitätsmedizin Berlin, Germany before (from March 1 to 26, 2020, n=30) and after (from April 4 to November 19, 2020, n=164) dexamethasone became standard of care. The study is registered in the German and the WHO International Clinical Trials Registry (DRKS00021688). Findings: The assay produces reproducible (median inter-batch CV of 10.9%) absolute quantification of 47 peptides with high sensitivity (median LLOQ of 143 ng/ml) and accuracy (median 96.8%). In both studies, the assay reproducibly captured hallmarks of COVID-19 infection and severity, as it distinguished healthy individuals, mild, moderate, and severe COVID-19. In the post-dexamethasone cohort, the assay predicted survival with an accuracy of 0.83 (108/130), and death with an accuracy of 0.76 (26/34) in the median 2.5 weeks before the outcome, thereby outperforming compound clinical risk assessments such as SOFA, APACHE II, and ABCS scores. Interpretation: Disease severity and clinical outcomes of patients with COVID-19 can be stratified and predicted by the routine-applicable panel assay that combines known and novel COVID-19 biomarkers. The prognostic value of this assay should be prospectively assessed in larger patient cohorts for future support of clinical decisions, including evaluation of sample flow in routine setting. The possibility to objectively classify COVID-19 severity can be helpful for monitoring of novel therapies, especially in early clinical trials. Funding: This research was funded in part by the European Research Council (ERC) under grant agreement ERC-SyG-2020 951475 (to M.R) and by the Wellcome Trust (IA 200829/Z/16/Z to M.R.). The work was further supported by the Ministry of Education and Research (BMBF) as part of the National Research Node 'Mass Spectrometry in Systems Medicine (MSCoresys)', under grant agreements 031L0220 and 161L0221. J.H. was supported by a Swiss National Science Foundation (SNSF) Postdoc Mobility fellowship (project number 191052). This study was further supported by the BMBF grant NaFoUniMedCOVID-19 - NUM-NAPKON, FKZ: 01KX2021. The study was co-funded by the UK's innovation agency, Innovate UK, under project numbers 75594 and 56328.

Activating transcription factor 4-dependent lactate dehydrogenase activation as a protective response to amyloid beta toxicity.

Accumulation of amyloid beta peptides is thought to initiate the pathogenesis of Alzheimer's disease. However, the precise mechanisms mediating their neurotoxicity are unclear. Our microarray analyses show that, in Drosophila models of amyloid beta 42 toxicity, genes involved in the unfolded protein response and metabolic processes are upregulated in brain. Comparison with the brain transcriptome of early-stage Alzheimer's patients revealed a common transcriptional signature, but with generally opposing directions of gene expression changes between flies and humans. Among these differentially regulated genes, lactate dehydrogenase (Ldh) was up-regulated by the greatest degree in amyloid beta 42 flies and the human orthologues (LDHA and LDHB) were down-regulated in patients. Functional analyses revealed that either over-expression or inhibition of Ldh by RNA interference (RNAi) slightly exacerbated climbing defects in both healthy and amyloid beta 42-induced Drosophila. This suggests that metabolic responses to lactate dehydrogenase must be finely-tuned, and that its observed upregulation following amyloid beta 42 production could potentially represent a compensatory protection to maintain pathway homeostasis in this model, with further manipulation leading to detrimental effects. The increased Ldh expression in amyloid beta 42 flies was regulated partially by unfolded protein response signalling, as ATF4 RNAi diminished the transcriptional response and enhanced amyloid beta 42-induced climbing phenotypes. Further functional studies are required to determine whether Ldh upregulation provides compensatory neuroprotection against amyloid beta 42-induced loss of activating transcription factor 4 activity and endoplasmatic reticulum stress. Our study thus reveals dysregulation of lactate dehydrogenase signalling in Drosophila models and patients with Alzheimer's disease, which may lead to a detrimental loss of metabolic homeostasis. Importantly, we observed that down-regulation of ATF4-dependent endoplasmic reticulum-stress signalling in this context appears to prevent Ldh compensation and to exacerbate amyloid beta 42-dependent neuronal toxicity. Our findings, therefore, suggest caution in the use of therapeutic strategies focussed on down-regulation of this pathway for the treatment of Alzheimer's disease, since its natural response to the toxic peptide may induce beneficial neuroprotective effects.

Mass Spectrometry Characterization of Higher Order Structural Changes Associated with the Fc-glycan Structure of the NISTmAb Reference Material, RM 8761.

As monoclonal antibodies (mAbs) rapidly emerge as a dominant class of therapeutics, so does the need for suitable analytical technologies to monitor for changes in protein higher order structure (HOS) of these biomolecules. Reference materials (RM) serve a key analytical purpose of benchmarking the suitability and robustness of both established and emerging analytical procedures for both drug producers and regulators. Here, two simple enzymatic protocols for generating Fc-glycan variants from the NISTmAb RM are described and both global and localized changes in HOS between the RM and these Fc-glycan variants are characterized using hydrogen deuterium exchange-mass spectrometry (HDX-MS) and ion mobility spectrometry-mass spectrometry (IMS-MS) measurements. An alternative statistical approach is described where measurement thresholds that differentiate between measurement variability and significant structural changes were established on the basis of experimental data. Measurements revealed decreases in structural stability correlating with the degree of Fc-glycan structure loss, especially at the CH2/CH3 domain interface. These data promote the use of this RM and these Fc-glycan variants for establishing the sensitivity of and validating analytical methods for the detection of HOS measurements of mAbs.

Dynamic changes in the brain protein interaction network correlates with progression of Aß42 pathology in Drosophila.

Alzheimer's disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. Here, we present a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aß42 gene. We identified 3093 proteins from flies that were induced to express Aß42 and age-matched healthy controls using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aß42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. Our unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems.

Assessment of Recovery of Milk Protein Allergens from Processed Food for Mass Spectrometry Quantification.

Assessing the recovery of food allergens from solid processed matrixes is one of the most difficult steps that needs to be overcome to enable the accurate quantification of protein allergens by immunoassay and MS. A feasibility study is described herein applying International System of Units (SI)-traceably quantified milk protein solutions to assess recovery by an improved extraction method. Untargeted MS analysis suggests that this novel extraction method can be further developed to provide high recoveries for a broad range of food allergens. A solution of α-casein was traceably quantified to the SI for the content of α-S1 casein. Cookie dough was prepared by spiking a known amount of the SI-traceable quantified solution into a mixture of flour, sugar, and soya spread, followed by baking. A novel method for the extraction of protein food allergens from solid matrixes based on proteolytic digestion was developed, and its performance was compared with the performance of methods reported in the literature.

Online Hydrogen-Deuterium Exchange Traveling Wave Ion Mobility Mass Spectrometry (HDX-IM-MS): a Systematic Evaluation.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an important tool for measuring and monitoring protein structure. A bottom-up approach to HDX-MS provides peptide level deuterium uptake values and a more refined localization of deuterium incorporation compared with global HDX-MS measurements. The degree of localization provided by HDX-MS is proportional to the number of peptides that can be identified and monitored across an exchange experiment. Ion mobility spectrometry (IMS) has been shown to improve MS-based peptide analysis of biological samples through increased separation capacity. The integration of IMS within HDX-MS workflows has been commercialized but presently its adoption has not been widespread. The potential benefits of IMS, therefore, have not yet been fully explored. We herein describe a comprehensive evaluation of traveling wave ion mobility integrated within an online-HDX-MS system and present the first reported example of UDMSE acquisition for HDX analysis. Instrument settings required for optimal peptide identifications are described and the effects of detector saturation due to peak compression are discussed. A model system is utilized to confirm the comparability of HDX-IM-MS and HDX-MS uptake values prior to an evaluation of the benefits of IMS at increasing sample complexity. Interestingly, MS and IM-MS acquisitions were found to identify distinct populations of peptides that were unique to the respective methods, a property that can be utilized to increase the spatial resolution of HDX-MS experiments by >60%. Graphical Abstract ᅟ.

Autophosphorylation activity of a soluble hexameric histidine kinase correlates with the shift in protein conformational equilibrium.

In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal "receiver domain" that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the "compact" form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG.