Search Results (55)

Nucleocytoplasmic trafficking is a highly regulated process that allows the cell to control the partitioning of proteins and nucleic acids between the cytosolic and nuclear compartments. The Ebola virus minor matrix protein VP24 (eVP24) hijacks this process by binding to a region on the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear importers. This region overlaps with the activated transcription factor STAT1 binding site on KPNAs, preventing STAT1 nuclear localization and activation of antiviral gene transcription. However, the molecular interactions of eVP24-KPNA5 binding that lead to the nuclear localization of eVP24 remain poorly characterized. Here, we show that trafficking of eVP24 into the nucleus by KPNA5 requires simultaneous binding of cargo. We also describe the conformational dynamics of KPNA5 and interactions with eVP24 and cargo nuclear localization sequences (NLS) using biophysical approaches. Our results reveal that eVP24 binding to KPNA5 does not impact cargo NLS binding to KPNA5, indicating that simultaneous binding of both cellular cargo and eVP24 to KPNA5 is likely required for nuclear trafficking. Together, these results provide a biophysical basis for how Ebola virus VP24 protein gains access to the nucleus during Ebola virus infection. Full article

Background/Objectives: An initial COVID-19 candidate vaccine containing a purified ancestral SARS-CoV-2 spike antigen was characterized with an ELISA using recombinant monoclonal antibodies (mAbs) generated against this variant. Upon the emergence of a new Beta (B.1.351) spike variant early in the pandemic, the assessment of a bivalent vaccine containing ancestral and Beta spike antigens began. Due to accelerated project timelines, mAbs generated specifically against the Beta spike antigen were not available at the time to address assay development and vaccine testing requirements. Methods: Using only the initial mAb panel raised against the ancestral spike antigen, an epitope-blocking ELISA strategy was developed to independently measure Beta spike antigen in bivalent vaccine formulations. To facilitate this, epitope profiling of spike antigens from both ancestral and Beta variants was performed with biolayer interferometry and hydrogen–deuterium exchange mass spectrometry using the original panel of mAbs. Results: The resulting blocking ELISA was precise and specific for the Beta spike antigen and detected the expected amount of this antigen in bivalent vaccine formulations. The specific amount of ancestral spike protein in the bivalent vaccine was also confirmed using the original ELISA developed at the onset of the pandemic. Conclusions: This epitope-blocking strategy helped to overcome key reagent availability issues and could be applied to other projects involving related proteins. Full article

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α₂β₂γ₂ heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α₂β₂γ₂ complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme. Full article

CNOT1, a key scaffold in the CCR4-NOT complex, plays a critical role in mRNA decay, particularly in the regulation of inflammatory responses through its interaction with tristetraprolin. A fragment of the middle part of CNOT1 (residues 800–999) is an example of an α-helical HEAT-like repeat domain. The HEAT motif is an evolutionarily conserved motif present in scaffolding and transport proteins across a wide range of organisms. Using hydrogen/deuterium exchange mass spectrometry (HDX MS), a method that has not been widely explored in the context of HEAT repeats, we analysed the structural dynamics of wild-type CNOT1(800–999) and its two double point mutants (E893A/Y900A, E893Q/Y900H) to find the individual contributions of these CNOT1 residues to the molecular recognition of tristetraprolin (TTP). Our results show that the differences in the interactions of CNOT1(800–999) variants with the TTP peptide fragment are due to the absence of the critical residues resulting from point mutations and not due to the perturbation of the protein structure. Nevertheless, the HDX MS was able to detect slight local changes in structural dynamics induced by protein point mutations, which are usually neglected in studies of intermolecular interactions. Full article

The main proteinase (Mpro), or 3CLpro, is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle and is responsible for breaking down and releasing vital functional viral proteins crucial for virus development and transmission. As a catalytically active dimer, its dimerization interface has become an attractive target for antiviral drug development. Recent research has extensively investigated the enzymatic activity of Mpro, focusing on its role in regulating the coronavirus replication complex and its significance in virus maturation and infectivity. Computational investigations have identified four druggable pockets, suggesting potential allosteric sites beyond the substrate-binding region. Empirical validation through site-directed alanine mutagenesis has targeted residues in both the active and allosteric regions and corroborated these predictions. Structural studies of drug target proteins can inform therapeutic approaches, with metadynamics simulations shedding light on the role of H163 in regulating Mpro function and providing insights into its dynamic equilibrium to the wild-type enzyme. Despite the efficacy of vaccines and drugs in mitigating SARS-CoV-2 spread, its ongoing viral evolution, selective pressures, and continued transmission pose challenges, potentially leading to resistant mutations. Phylogenetic analyses have indicated the existence of several resistant variations predating drug introduction to the human population, emphasizing the likelihood of drug spread. Hydrogen/deuterium-exchange mass spectrometry reveals the structural influence of the mutation. At the same time, clinical trials on 3CLPro inhibitors underscore the clinical significance of reduced enzymatic activity and offer avenues for future therapeutic exploration. Understanding the implications of 3CLPro mutations holds promise for shaping forthcoming therapeutic strategies against COVID-19. This review delves into factors influencing mutation rates and identifies areas warranting further investigation, providing a comprehensive overview of Mpro mutations, categorization, and terminology. Moreover, we examine their associations with clinical outcomes, illness severity, unresolved issues, and future research prospects, including their impact on vaccine efficacy and potential therapeutic targeting. Full article

DJ-1 is a vital enzyme involved in the maintenance of mitochondrial health, and its mutation has been associated with an increased risk of Parkinson’s disease (PD). Effective regulation of DJ-1 activity is essential for the well-being of mitochondria, and DJ-1 is thus a potential target for PD drug development. In this study, two peptides (¹⁵EEMETIIPVDVMRRA²⁹ and ⁴⁷SRDVVICPDA⁵⁶) were utilized with the aim of enhancing the activity of DJ-1. The mechanisms underlying the activity enhancement by these two peptides were investigated using hydrogen/deuterium exchange mass spectrometry (HDXMS). The HDXMS results revealed distinct mechanisms. Peptide 1 obstructs the access of solvent to the dimer interface and stabilizes the α/β hydrolase structure, facilitating substrate binding to a stabilized active site. Conversely, peptide 2 induces a destabilization of the α/β hydrolase core, enhancing substrate accessibility and subsequently increasing DJ-1 activity. The binding of these two peptides optimizes the activity site within the dimeric structure. These findings offer valuable insights into the mechanisms underlying the activity enhancement of DJ-1 by the two peptides, potentially aiding the development of new drugs that can enhance the activity of DJ-1 and, consequently, advance PD treatment. Full article

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF₂ slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N₂ gas saturated with ²H₂O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm². Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses. Full article

Tick Anticoagulant Peptide (TAP), a 60-amino acid protein from the soft tick Ornithodoros moubata, inhibits activated coagulation factor X (fXa) with almost absolute specificity. Despite TAP and Bovine Pancreatic Trypsin Inhibitor (BPTI) (i.e., the prototype of the Kunitz-type protease inhibitors) sharing a similar 3D fold and disulphide bond topology, they have remarkably different amino acid sequence (only ~24% sequence identity), thermal stability, folding pathways, protease specificity, and even mechanism of protease inhibition. Here, fully active and correctly folded TAP was produced in reasonably high yields (~20%) by solid-phase peptide chemical synthesis and thoroughly characterised with respect to its chemical identity, disulphide pairing, folding kinetics, conformational dynamics, and fXa inhibition. The versatility of the chemical synthesis was exploited to perform structure–activity relationship studies on TAP by incorporating non-coded amino acids at positions 1 and 3 of the inhibitor. Using Hydrogen–Deuterium Exchange Mass Spectrometry, we found that TAP has a remarkably higher conformational flexibility compared to BPTI, and propose that these different dynamics could impact the different folding pathway and inhibition mechanisms of TAP and BPTI. Hence, the TAP/BPTI pair represents a nice example of divergent evolution, while the relative facility of TAP synthesis could represent a good starting point to design novel synthetic analogues with improved pharmacological profiles. Full article

The purpose of this study was to develop a formulation for a recombinant prefusion spike protein vaccine against SARS-CoV-2. It was found that the spike protein was susceptible to aggregation due to mechanical stress. Therefore, formulation studies were initiated focused on screening pharmaceutical excipients capable of preventing this. The screening of a panel of potential stabilizing conditions found that Tween 20 could inhibit mechanically induced aggregation. A concentration-dependent study indicated that a higher concentration of Tween 20 (0.2% v/v) was required to prevent conformational changes in the trimer. The conformational changes induced by mechanical stress were characterized by size exclusion chromatography (SEC) and hydrogen–deuterium exchange mass spectrometry (HDX-MS), indicating the formation of an extended trimeric conformation that was also unable to bind to antibodies directed to the S2 domain. Long-term stability modeling, using advanced kinetic analysis, indicated that the formulation containing 0.2% (v/v) Tween 20 at a neutral pH was predicted to be stable for at least two years at 2 °C to 8 °C. Additional stabilizer screening conducted by thermal shift assay indicated that sucrose and glycerol were able to significantly increase the spike protein melting temperature (Tm) and improve the overall thermostability of the spike protein in a short-term stability study. Thus, while 0.2% (v/v) Tween 20 was sufficient to prevent aggregation and to maintain spike protein stability under refrigeration, the addition of sucrose further improved vaccine thermostability. Altogether, our study provides a systematic approach to the formulation of protein-based COVID-19 vaccine and highlights the impact of mechanical stress on the conformation of the spike protein and the significance of surfactants and stabilizers in maintaining the structural and functional integrity of the spike protein. Full article

Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody’s surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography. Full article

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange–mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment. Full article

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid. Full article

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen–antibody and other protein–protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated. Full article

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time–m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D₂O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N¹-acetylspermidine. Full article

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca²⁺ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and identified that ⁹¹QEKAHIAEHMDVPIDTSKMSEQELQFHY¹¹⁸ from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV. Full article