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Phosphatidylinositol 4-kinase alpha (PI4KA) maintains the phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools of the plasma membrane. A key regulator of PI4KA is its association into a complex with TTC7 and FAM126 proteins. This complex can be regulated by the CNAβ1 isoform of the phosphatase calcineurin. We previously identified that CNAβ1 directly binds to FAM126A. Here, we report a cryoelectron microscopic (cryo-EM) structure of a truncated PI4KA complex bound to calcineurin, revealing a unique direct interaction between PI4KA and calcineurin. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and computational analysis show that calcineurin forms a complex with an evolutionarily conserved IKISVT sequence in PI4KA's horn domain. We also characterized conserved LTLT and PSISIT calcineurin binding sequences in the C terminus of FAM126A. These dual sites in PI4KA and FAM126A are both in close proximity to phosphorylation sites in the PI4KA complex, suggesting key roles of calcineurin-regulated phosphosites in PI4KA regulation. This work reveals novel insight into how calcineurin can regulate PI4KA activity. [Display omitted] • 3.50 Å cryo-EM structure of the PI4KA complex bound to calcineurin • calcineurin interacts with the solenoid and dimerization domains of PI4KA • calcineurin engages with the disordered C-terminus of FAM126A Dysregulation of PI4KA is involved in human disease; however, the role of interacting kinases/phosphatases in its regulation is unknown. Here, Shaw et al. show a direct interaction of the phosphatase calcineurin with the PI4KA complex at two unique sites, providing molecular insight into how PI4KA is regulated. [ABSTRACT FROM AUTHOR]

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin pre-activation. We compare the conformational changes of β-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and βXX to transform β-arrestin-2 into the pre-active state. Synopsis: The mechanism of β-arrestin (βarr2) pre-activation by PIP2 has not yet been fully understood. This study suggests that PIP2 induces allosteric conformational changes in βarr2, which ultimately affect the conformational dynamics of the βXX strand. PIP2 increases the conformational dynamics of the βXX strand, indicating the pre-activation of βarr2. HDX-MS and mutational studies have revealed the allosteric structural pathway from the PIP2-binding sites to the βXX strand. Conformational perturbation of the back loop through the gate loop of βarr2 is the allosteric structural pathway of the PIP2-induced pre-activation of βarr2. The mechanism of β-arrestin (βarr2) pre-activation by PIP2 has not yet been fully understood. This study suggests that PIP2 induces allosteric conformational changes in βarr2, which ultimately affect the conformational dynamics of the βXX strand. [ABSTRACT FROM AUTHOR]

The gene PIK3CA, encoding the catalytic subunit p110α of PI3Kα, is the second most frequently mutated gene in cancer, with the highest frequency oncogenic mutants occurring in the C-terminus of the kinase domain. The C-terminus has a dual function in regulating the kinase, playing a putative auto-inhibitory role for kinase activity and being absolutely essential for binding to the cell membrane. However, the molecular mechanisms by which these C-terminal oncogenic mutations cause PI3Kα overactivation remain unclear. To understand how a spectrum of C-terminal mutations of PI3Kα alter kinase activity compared to the WT, we perform unbiased and biased Molecular Dynamics simulations of several C-terminal mutants and report the free energy landscapes for the C-terminal “closed-to-open” transition in the WT, H1047R, G1049R, M1043L and N1068KLKR mutants. Results are consistent with HDX-MS experimental data and provide a molecular explanation why H1047R and G1049R reorient the C-terminus with a different mechanism compared to the WT and M1043L and N1068KLKR mutants. Moreover, we show that in the H1047R mutant, the cavity, where the allosteric ligands STX-478 and RLY-2608 bind, is more accessible contrary to the WT. This study provides insights into the molecular mechanisms underlying activation of oncogenic PI3Kα by C-terminal mutations and represents a valuable resource for continued efforts in the development of mutant selective inhibitors as therapeutics.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon β-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of β-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high‐resolution hydrogen‐deuterium exchange mass spectrometry (HDX‐MS), an emerging and powerful biophysical technique. Using HDX‐MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo‐ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX‐MS maps for PTP1B with active‐site versus allosteric small‐molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active‐site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX‐MS for elucidating aspects of protein conformational dynamics and allosteric effects of small‐molecule ligands and highlights the potential of integrating HDX‐MS alongside other complementary methods, such as room‐temperature X‐ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics. [ABSTRACT FROM AUTHOR]

The lipolytic processing of triglyceride-rich lipoproteins (TRLs) by lipoprotein lipase (LPL) is crucial for the delivery of dietary lipids to the heart, skeletal muscle, and adipose tissue. The processing of TRLs by LPL is regulated in a tissue-specific manner by a complex interplay between activators and inhibitors. Angiopoietin-like protein 4 (ANGPTL4) inhibits LPL by reducing its thermal stability and catalyzing the irreversible unfolding of LPLs α/β-hydrolase domain. We previously mapped the ANGPTL4 binding site on LPL and defined the downstream unfolding events resulting in LPL inactivation. The binding of LPL to glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 protects against LPL unfolding. The binding site on LPL for an activating cofactor, apolipoprotein C2 (APOC2), and the mechanisms by which APOC2 activates LPL have been unclear and controversial. Using hydrogen-deuterium exchange/mass spectrometry, we now show that APOC2s C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2s binding site on LPL overlaps with that for ANGPTL4, but their effects on LPL conformation are distinct. In contrast to ANGPTL4, APOC2 increases the thermal stability of LPL and protects it from unfolding. Also, the regions of LPL that anchor the lid are stabilized by APOC2 but destabilized by ANGPTL4, providing a plausible explanation for why APOC2 is an activator of LPL, while ANGPTL4 is an inhibitor. Our studies provide fresh insights into the molecular mechanisms by which APOC2 binds and stabilizes LPL-and properties that we suspect are relevant to the conformational gating of LPLs active site.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Methionine oxidation can substantially alter the structure and functionality of monoclonal antibodies (mAbs), especially when it occurs in the complementarity-determining regions (CDRs). It is imperative to fully understand the effects of methionine oxidation because these modifications can affect the binding affinity, stability, and immunogenicity of mAbs. Moreover, the presence of multiple methionines in close proximity within the amino acid sequence increases the complexity of accurate characterization, and sophisticated analytical methods are required to detect these modifications. In this study, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) and homology modeling to investigate the effects of dual methionine oxidations (heavy chain (HC) Met111 and Met115) within a single CDR on the structure of a mAb. Our findings reveal that the solvent-accessible methionine (HC Met111) is more prone to oxidation, but such a modification does not result in conformational changes in the mAb. In contrast, the methionine (HC Met115) at the VH-VL interface, when subjected to different oxidative stresses, can undergo oxidation with selective stereochemistry. This can lead to predominant formation of either the S- or R-form of methionine sulfoxide diastereomer, each of which can induce distinct local conformational changes. A mechanism is proposed to elucidate these observations in this particular antibody. Furthermore, binding assays confirm that both CDR methionine oxidations do not compromise antigen binding, which alleviates concerns about potential loss of therapeutic efficacy.

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3‐dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX‐MS experiments show an overall decrease in H‐bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX‐MS results with the predicted GFAP‐dimer structure by AlphaFold‐Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices. [ABSTRACT FROM AUTHOR]

Hirudin from Hirudo medicinalis is a bivalent α‐Thrombin (αT) inhibitor, targeting the enzyme active site and exosite‐I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land‐living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite‐II and therefore (besides the free protease) it also blocks thrombomodulin‐bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild‐type haemadin and the oxidation resistant Met5 → nor‐Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site‐directed mutagenesis, spectroscopic, ligand‐displacement, and Hydrogen/Deuterium Exchange‐Mass Spectrometry techniques to map the αT regions relevant for the interaction with full‐length haemadin and with the synthetic N‐ and C‐terminal peptides Haem(1–10) and Haem(45–57). Haem(1–10) competitively binds to/inhibits αT active site (KI = 1.9 μM) and its potency was enhanced by 10‐fold after Phe3 → β‐Naphthylalanine exchange. Conversely to full‐length haemadin, haem(45–57) displays intrinsic affinity for exosite‐I (KD = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1–9 and 45–57 were joined together through a 3‐Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite‐I. Haemanorm can be regarded as a novel class of hirulog‐like αT inhibitors with potential pharmacological applications. [ABSTRACT FROM AUTHOR]

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.

ABSTRACTDevelopment of novel bispecific antibody (bsAb) platforms offers unprecedented opportunities for a wide variety of therapeutic applications. However, the expression and manufacturing of bsAbs with desired structures can be challenging. Owing to the uniqueness of each bsAb platform, more comprehensive and customized structural characterization is particularly important to understand the chemical or biological reactivity of bsAbs, as well as to guide process development, risk assessment, and manufacturing. In this work, we performed higher order structure characterization of the Regeneron bsAb platform with Fc site-specific substitutions through hydrogen deuterium exchange mass spectrometry (HDX-MS). Structural deprotection was identified at the CH2-CH3 interface in the Fc domain, owing to the site-specific substitutions. The structural deprotection was found to correlate with the decreased conformational stability of Fc domain. Under oxidative and thermal stress conditions, the Met residues located near the structurally deprotected region were identified to be susceptible to oxidation. In addition, the introduction of substitutions in the bsAb Fc resulted in a slight reduction of its binding affinity to the neonatal Fc receptor (FcRn). The detailed structural elucidation by HDX-MS improves understanding of the structure–property relationship of the Regeneron bsAb format, thus greatly aiding in process development, risk assessment, and manufacturing.

ABSTRACTHageman factor (FXII) is an essential component in the intrinsic coagulation cascade and a therapeutic target for the prophylactic treatment of hereditary angioedema (HAE). CSL312 (garadacimab) is a novel high-affinity human antibody capable of blocking activated FXII activity that is currently undergoing Phase 3 clinical trials in HAE. Structural studies using hydrogen/deuterium exchange coupled to mass spectrometry revealed evidence of interaction between the antibody and regions surrounding the S1 specificity pocket of FXII, including the 99-loop, 140-loop, 180-loop, and neighboring regions. We propose complementarity-determining regions (CDRs) in heavy-chain CDR2 and CDR3 as potential paratopes on garadacimab, and the 99-loop, 140-loop, 180-loop, and 220-loop as binding sites on the beta chain of activated FXII (β-FXIIa).

Background: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions. Methods: Visible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX). Results: Particles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying. Discussion: The spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process. [ABSTRACT FROM AUTHOR]

The complex between lipoprotein lipase (LPL) and its endothelial receptor (GPIHBP1) is responsible for the lipolytic processing of triglyceride-rich lipoproteins (TRLs) along the capillary lumen, a physiologic process that releases lipid nutrients for vital organs such as heart and skeletal muscle. LPL activity is regulated in a tissue-specific manner by endogenous inhibitors (angiopoietin-like [ANGPTL] proteins 3, 4, and 8), but the molecular mechanisms are incompletely understood. ANGPTL4 catalyzes the inactivation of LPL monomers by triggering the irreversible unfolding of LPL's α/β-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including β2, β3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on β3-α3 and progress to β5 and β4-α4, ultimately leading to the irreversible unfolding of regions that form LPL's catalytic pocket. LPL unfolding is context dependent and varies with the thermal stability of LPL's α/β-hydrolase domain (Tm of 34.8 °C). GPIHBP1 binding dramatically increases LPL stability (Tm of 57.6 °C), while ANGPTL4 lowers the onset of LPL unfolding by ∼20 °C, both for LPL and LPL•GPIHBP1 complexes. These observations explain why the binding of GPIHBP1 to LPL retards the kinetics of ANGPTL4-mediated LPL inactivation at 37 °C but does not fully suppress inactivation. The allosteric mechanism by which ANGPTL4 catalyzes the irreversible unfolding and inactivation of LPL is an unprecedented pathway for regulating intravascular lipid metabolism.

ABSTRACT Multiple respiratory viruses, including influenza A virus (IAV), can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. Indoors, small exhaled aerosols undergo rapid acidification to pH ~4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes; however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Protein changes were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 s post-exposure to acidic conditions. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques, such as X-ray crystallography, highlighting the ease and usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Additionally, virion integrity was partially but irreversibly affected by acidic conditions, with a progressive unfolding of the internal matrix protein 1 (M1) that aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid the development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future. IMPORTANCE It is well established that COVID-19, influenza, and many other respiratory diseases can be transmitted by the inhalation of aerosolized viruses. Many studies have shown that the survival time of these airborne viruses is limited, but it remains an open question as to what drives their infectivity loss. Here, we address this question for influenza A virus by investigating structural protein changes incurred by the virus under conditions relevant to respiratory aerosol particles. From prior work, we know that expelled aerosols can become highly acidic due to equilibration with indoor room air, and our results indicate that two viral proteins are affected by these acidic conditions at multiple sites, leading to virus inactivation. Our findings suggest that the development of air treatments to quicken the speed of aerosol acidification would be a major strategy to control infectious bioburdens in the air.

Background: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. Methods: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. Results: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. Conclusions: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing. [ABSTRACT FROM AUTHOR]

The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) has become widely popular for mapping protein-ligand interfaces, for understanding protein-protein interactions, and for discovering dynamic allostery. Several platforms are now available which provide large data sets of amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data. Although many of these platforms provide some down-stream processing, a comprehensive software that provides the most commonly used down-stream processing tools such as automatic back-exchange correction options, analysis of overlapping peptides, calculations of relative deuterium uptake into regions of the protein after such corrections, rigorous statistical analysis of the significance of uptake differences, and generation of high quality figures for data presentation is not yet available. Here we describe the Deuterium Exchange Correction and Analysis (DECA) software package, which provides all these downstream processing options for data from the most popular mass spectrometry platforms. The major functions of the software are demonstrated on sample data.

PI3Kγ is a critical immune signaling enzyme activated downstream of diverse cell surface molecules, including Ras, PKCβ activated by the IgE receptor, and Gβγ subunits released from activated GPCRs. PI3Kγ can form two distinct complexes, with the p110γ catalytic subunit binding to either a p101 or p84 regulatory subunit, with these complexes being differentially activated by upstream stimuli. Here, using a combination of cryo electron microscopy, HDX-MS, and biochemical assays, we have identified novel roles of the helical domain of p110γ in regulating lipid kinase activity of distinct PI3Kγ complexes. We defined the molecular basis for how an allosteric inhibitory nanobody potently inhibits kinase activity through rigidifying the helical domain and regulatory motif of the kinase domain. The nanobody did not block either p110γ membrane recruitment or Ras/Gβγ binding, but instead decreased ATP turnover. We also identified that p110γ can be activated by dual PKCβ helical domain phosphorylation leading to partial unfolding of an N-terminal region of the helical domain. PKCβ phosphorylation is selective for p110γ-p84 compared to p110γ-p101, driven by differential dynamics of the helical domain of these different complexes. Nanobody binding prevented PKCβ-mediated phosphorylation. Overall, this work shows an unexpected allosteric regulatory role of the helical domain of p110γ that is distinct between p110γ-p84 and p110γ-p101 and reveals how this can be modulated by either phosphorylation or allosteric inhibitory binding partners. This opens possibilities of future allosteric inhibitor development for therapeutic intervention.

Secretory preproteins of the Sec pathway are targeted post‐translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non‐folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl‐prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen–deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near‐residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a β‐turn in the earliest foldon and by signal peptide‐mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced in vivo secretion. These structural adaptations in a secretory protein facilitate trafficking. Synopsis: In bacteria, secreted proteins remain in a non‐folded state, which enhances their secretion. This study shows that such delayed folding is achieved by signal peptides and mature domain features that slow down the formation of discrete folding units (foldons) and disrupt the foldon pathway hierarchy. Periplasmic PpiA displays delayed folding compared to its fast‐folding structurally similar cytoplasmic homologue PpiB through a distinct, differently ordered hierarchical foldon pathway.A few grafted residues that affect highly stabilized native contacts can interconvert PpiB/PpiA folding kinetics and subsequent secretability.Signal peptides quickly acquire partial secondary structure, increase flexibility in succeeding regions of the mature domain and disturb initial foldon formation to promote secretion. [ABSTRACT FROM AUTHOR]

The endothelin receptor type B (ET B) exhibits promiscuous coupling with various heterotrimeric G protein subtypes including Gs, Gi/o, Gq/11, and G12/13. Recent fluorescence and structural studies have raised questions regarding the coupling efficiencies and determinants of these G protein subtypes. Herein, by utilizing an integrative approach, combining hydrogen/deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cellular systems, we investigated conformational changes of Gs, Gi, and Gq triggered by ET B activation. ET B coupled to Gi and Gq but not with Gs. We underscored the critical roles of specific regions, including the C terminus of Gα and intracellular loop 2 (ICL2) of ET B in ET B -Gi1 or ET B -Gq coupling. Although The C terminus of Gα is essential for ET B -Gi1 and ET B -Gq coupling, ET B ICL2 influences Gq-coupling but not Gi1-coupling. Our results suggest a differential coupling efficiency of ET B with Gs, Gi1, and Gq, accompanied by distinct conformational changes in G proteins upon ET B -induced activation. [Display omitted] • ET B couples with Gi and Gq, but not with Gs • ET B 's ICL2 affects Gq-coupling, not Gi1-coupling • A differential coupling efficiency of ET B with G protein subtypes is observed Ham et al. explored how the endothelin receptor type B (ET B) interacts with various G protein subtypes. They found that ET B couples with Gi and Gq, but not Gs, revealing key regions influencing this interaction. These findings highlight distinct coupling efficiencies and conformational changes in G proteins triggered by ET B. [ABSTRACT FROM AUTHOR]

Activated FXII (FXIIa) is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of hereditary angioedema (HAE). Here, we describe a high-resolution cryoelectron microscopy (cryo-EM) structure of the beta-chain from FXIIa (βFXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to βFXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This structural mechanism is likely the primary contributor to the inhibition of activated FXIIa proteolytic activity in HAE. Garadacimab Fab-βFXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXIIa. Structural analysis with other bona fide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a surprisingly similar mechanism of βFXIIa inhibition by garadacimab. In summary, the garadacimab Fab-βFXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes. [Display omitted] • Structure of therapeutic antibody garadacimab's Fab fragment complexed with βFXIIa • CDR-H3 insertion into the catalytic cleft of βFXIIa inhibits proteolytic activity • Structural basis for the high-affinity binding of garadacimab to βFXIIa is revealed • Garadacimab employs an inhibition mechanism similar to endogenous inhibitor C1-INH Drulyte et al. determined the high-resolution cryo-EM structure of the Fab fragment of garadacimab in complex with the beta-chain from activated FXIIa (βFXIIa). The structure reveals the molecular interactions responsible for their high-affinity binding and provides insights into the mechanism of proteolytic inhibition. [ABSTRACT FROM AUTHOR]

Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using β-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of β-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state. [Display omitted] • The roles of specific residues in the gate loop for β-arrestin activation are analyzed • K294 is essential for V2Rpp interaction, without affecting the active conformation • K292 and H295 are important for maintaining the stability of the basal state Kim et al. explored the roles of specific residues (K292, K294, and H295) in the gate loop for β-arrestin-1 activation. They elucidated that while K294 is crucial for V2Rpp interaction, K292 and H295 help stabilize the polar core in the basal state. [ABSTRACT FROM AUTHOR]

Nicotinamide nucleotide transhydrogenases are integral membrane proteins that utilizes the proton motive force to reduce NADP+ to NADPH while converting NADH to NAD+. Atomic structures of various transhydrogenases in different ligand-bound states have become available, and it is clear that the molecular mechanism involves major conformational changes. Here we utilized hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map ligand binding sites and analyzed the structural dynamics of E. coli transhydrogenase. We found different allosteric effects on the protein depending on the bound ligand (NAD+, NADH, NADP+, NADPH). The binding of either NADP+ or NADPH to domain III had pronounced effects on the transmembrane helices comprising the proton-conducting channel in domain II. We also made use of cyclic ion mobility separation mass spectrometry (cyclic IMS-MS) to maximize coverage and sensitivity in the transmembrane domain, showing for the first time that this technique can be used for HDX-MS studies. Using cyclic IMS-MS, we increased sequence coverage from 68 % to 73 % in the transmembrane segments. Taken together, our results provide important new insights into the transhydrogenase reaction cycle and demonstrate the benefit of this new technique for HDX-MS to study ligand binding and conformational dynamics in membrane proteins.

Nonribosomal peptide synthetases (NRPSs) employ multiple domains, specifically arranged in modules, for the assembly‐line biosynthesis of a plethora of bioactive peptides. It is poorly understood how catalysis is correlated with the domain interplay and associated conformational changes. We developed FRET sensors of an elongation module to study in solution the intramodular interactions of the peptidyl carrier protein (PCP) with adenylation (A) and condensation (C) domains. Backed by HDX‐MS analysis, we discovered dynamic mixtures of conformations that undergo distinct population changes in favor of the PCP‐A and PCP‐C interactions upon completion of the adenylation and thiolation reactions, respectively. To probe this model we blocked PCP binding to the C domain by photocaging and triggered peptide bond formation with light. Changing intramodular domain affinities of the PCP appear to result in conformational shifts according to the logic of the templated assembly process. [ABSTRACT FROM AUTHOR]

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10 , a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis. [ABSTRACT FROM AUTHOR]

Simple Summary: In the present study, we addressed the unmet need for a molecular antibody (mAb) with high affinity and specificity against a truncated hyperactive isoform of human epidermal growth factor receptor 2 (HER2), called 611-carboxy terminal fragment (CTF)-p95HER2. Patients with p95HER2+ breast cancer are at risk of developing metastatic breast cancer with a poor prognosis and resistance to therapies targeting full-length HER2. We have generated a mAb named Oslo-2, which react specifically with 611-CTF-p95HER2 and has a high affinity. We also characterized the antigenic determinant (epitope) on the p95HER2 protein and the antigen-binding site (paratope) on the Oslo-2 mAb. The antibody can be used to develop antibody- or cell-based therapies targeting p95HER2, as well as a diagnostic assay to identify p95HER2+ disease. The expression of human epidermal growth factor receptor 2 (HER2) is a key classification factor in breast cancer. Many breast cancers express isoforms of HER2 with truncated carboxy-terminal fragments (CTF), collectively known as p95HER2. A common p95HER2 isoform, 611-CTF, is a biomarker for aggressive disease and confers resistance to therapy. Contrary to full-length HER2, 611-p95HER2 has negligible normal tissue expression. There is currently no approved diagnostic assay to identify this subgroup and no therapy targeting this mechanism of tumor escape. The purpose of this study was to develop a monoclonal antibody (mAb) against 611-CTF-p95HER2. Hybridomas were generated from rats immunized with cells expressing 611-CTF. A hybridoma producing a highly specific Ab was identified and cloned further as a mAb. This mAb, called Oslo-2, gave strong staining for 611-CTF and no binding to full-length HER2, as assessed in cell lines and tissues by flow cytometry, immunohistochemistry and immunofluorescence. No cross-reactivity against HER2 negative controls was detected. Surface plasmon resonance analysis demonstrated a high binding affinity (equilibrium dissociation constant 2 nM). The target epitope was identified at the N-terminal end, using experimental alanine scanning. Further, the mAb paratope was identified and characterized with hydrogen-deuterium-exchange, and a molecular model for the (Oslo-2 mAb:611-CTF-p95HER2) complex was generated by an experimental-information-driven docking approach. We conclude that the Oslo-2 mAb has a high affinity and is highly specific for 611-CTF-p95HER2. The Ab may be used to develop potent and safe therapies, overcoming p95HER2-mediated tumor escape, as well as for developing diagnostic assays. [ABSTRACT FROM AUTHOR]

Hydrogen-Deuterium exchange mass spectrometry's (HDX-MS) utility in identifying and characterizing protein-small molecule interaction sites has been established. The regions that are seen to be protected from exchange upon ligand binding indicate regions that may be interacting with the ligand, giving a qualitative understanding of the ligand binding pocket. However, quantitatively deriving an accurate high-resolution structure of the protein-ligand complex from the HDX-MS data remains a challenge, often limiting its use in applications such as small molecule drug design. Recent efforts have focused on the development of methods to quantitatively model Hydrogen-Deuterium exchange (HDX) data from computationally modeled structures to garner atomic level insights from peptide-level resolution HDX-MS. One such method, HDX ensemble reweighting (HDXer), employs maximum entropy reweighting of simulated HDX data to experimental HDX-MS to model structural ensembles. In this study, we implement and validate a workflow which quantitatively leverages HDX-MS data to accurately model protein-small molecule ligand interactions. To that end, we employ a strategy combining computational protein-ligand docking, molecular dynamics simulations, HDXer, and dimensional reduction and clustering approaches to extract high-resolution drug binding poses that most accurately conform with HDX-MS data. We apply this workflow to model the interaction of ERK2 and FosA with small molecule compounds and inhibitors they are known to bind. In five out of six of the protein-ligand pairs tested, the HDX derived protein-ligand complexes result in a ligand root-mean-square deviation (RMSD) within 2.5 Å of the known crystal structure ligand.

Hydrogen deuterium-exchange mass spectrometry (HDX-MS) is commonly used in the study of protein dynamics and protein interactions. By measuring the isotopic exchange of backbone amide hydrogens in solution, HDX-MS offers valuable structural insights into challenging biological systems. Traditional HDX-MS approaches utilize bottom-up (BU) proteomics, in which deuterated proteins are digested before MS analysis. BU-HDX enables the characterization of proteins with various sizes in simple protein mixtures or complex biological samples such as cell lysates. However, BU methods are inherently limited by the inability to resolve protein sub-populations arising from different protein conformations, such as those arising from post-translational modifications (PTMs). Alternatively, top-down (TD) HDX-MS detects the global deuterium uptake at the intact proteoform level, allowing direct probing of structural changes due to protein-protein interactions, PTMs, or conformational changes. Combining TD-HDX-MS with electron-based fragmentation techniques, such as electron capture dissociation (ECD) and electron transfer dissociation (ETD), has demonstrated the feasibility of studying intact protein interactions with amino acid-level resolution. Here, we present a brief overview of methodologies, limitations, and applications of TD-HDX-MS using direct infusion techniques and LC-based approaches. Furthermore, we conclude with a perspective on the future directions for TD-HDX-MS., (© 2024 John Wiley & Sons Ltd.)

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre--steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-50 -phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction. [ABSTRACT FROM AUTHOR]

Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal α-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes.

Bak is a pore-forming Bcl2 protein that induces apoptosis at the outer mitochondrial membrane, which can either proceed via Bak oligomerization or be inhibited by anti-apoptotic Bcl2 proteins, such as BclxL. BclxL is very efficient in inhibiting Bak pore formation, but the mechanistic basis of this preferred interaction has remained enigmatic. Here, we identify Bakα1 as a second binding site for BclxL and show that it specifically interacts with the Bcl2-homology (BH)3 binding groove of BclxL. The affinity between BclxL and Bakα1 is weaker than with Bak-BH3, suggesting that Bakα1, being exposed early in the pore-forming trajectory, transiently captures BclxL, which subsequently transitions to the proximal BH3 site. Bak variants where the initial transient interaction with BclxL is modulated show a markedly altered response to BclxL inhibition. This work contributes to a better mechanistic understanding of the fine-tuned interactions between different players of the Bcl2 protein family. [Display omitted] • Bakα1 binds to the BH3 binding groove of BclxL with micromolar affinity • Bakα1 forms an encounter complex with BclxL in the early stages of Bak activation • The interplay of Bakα1 and BH3 increases the efficiency of inhibition by BclxL Leitl et al. report on mechanistic details of the inhibition of the pro-apoptotic effector Bcl2 protein Bak by BclxL. Through structural and biophysical investigations, they uncover a second binding site for BclxL on Bak, suggesting a two-step binding model that ensures efficient inhibition of Bak to prevent unwanted pore formation. [ABSTRACT FROM AUTHOR]

C-Reactive protein (CRP) is an important marker for in vitro diagnosis (IVD) of inflammation. However, CRP immunoturbidimetric kits from different manufacturers exhibit inconsistency in evaluation, making clinical diagnosis challenging. The use of immunological methods in diagnosis means that the differences in epitopes across kits may directly lead to inconsistent results. Therefore, to provide consistent results, it is essential to perform epitope mapping of different kits. The composition of antibodies in a single kit is typically complex, with a combination of polyclonal antibodies or monoclonal antibodies. Here, we show an epitope screening strategy for complex antibodies in a kit based on hydrogen-deuterium exchange mass spectrometry (HDX-MS). We applied this workflow to successfully map the epitopes for three kits from three different manufacturers and compared their quantitative results. We obtained different quantitative results using kits from different manufacturers upon epitope mapping, confirming the correlation between the quantitative results and the epitopes. Thus, we have established a workflow based on HDX-MS to screen epitopes in IVD kits. This work helps determine the quantitative accuracy of a kit based on structural information, can guide the design and production of IVD reagents, and further improves the accuracy of IVD. [ABSTRACT FROM AUTHOR]

[Display omitted] Lipases are widely used in the detergent industry and must withstand harsh conditions involving both anionic and zwitterionic surfactants at alkaline pH. Thermomyces lanuginosus lipase (TlL) is often used and stays active at high concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) at pH 8.0, but is sensitive to SDS at pH 6.0 and below. We propose that enhanced stability at pH 8.0 results from a structurally distinct complex formation with SDS. We use small-angle X-ray scattering (SAXS) to elucidate structures of TlL:SDS at pH 4.0, 6.0, and 8.0 and further investigate the complexes at pH 8.0 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). At pH 4.0, large dense aggregates are formed at low [SDS], which become gradually less dense at higher [SDS], resulting in a core–shell structure. At pH 6.0, SDS induces a TlL dimer and forms a hemi-micelle along the side of the dimer. At higher [SDS], TlL adopts a core–shell structure. At pH 8.0, TlL forms a dimer with a SDS hemi-micelle but avoids a core–shell structure and maintains activity. Three helices are identified as SDS anchor points. This study provides important structural insight into the stability of TlL towards SDS under alkaline conditions. [ABSTRACT FROM AUTHOR]

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab. [ABSTRACT FROM AUTHOR]

Different models of protein folding favor different mechanisms. Some models rely on a defined pathway, while other models rely on a more heterogeneous set of pathways. The Foldon Hypothesis is based on the concept of a defined pathway. According to the Foldon Hypothesis, protein folding is characterized by the stepwise assembly of small, cooperative units called foldons. The Maximum Caliber (Max Cal) method provides an opportunity to test this model. The Max Cal method gives the probabilities for dynamical trajectories, in much the same way that the maximum entropy principle gives the probabilities for equilibrium states. In this study, the Max Cal method was applied to folding data for the protein cytochrome c (cyt c; Hu W, Kan ZY, Mayne L, Englander SW. Proc Natl Acad Sci U S A. 2016;113:3809–3814). The overall picture to emerge from this analysis is that the data do not require a dominant, defined pathway. The folding of cytochrome c is likely a heterogeneous process that includes multiple pathways. [ABSTRACT FROM AUTHOR]

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function, and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82 to 92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners and dictate its role in germ cell differentiation. [ABSTRACT FROM AUTHOR]

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

SUMMARY: Lipid anchors are common post‐translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C‐termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP–Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to describe the structural basis of plant REP–Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule‐specific, and in other cases they are common for a subset of Rabs. In particular, the C‐terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C‐terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast. Significance Statement: The Rab escort protein (REP) is part of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. We show by biochemical, biophysical, and genetic methods that the mechanism of action of REP, in particular the role of its C‐terminus in Rab binding and prenylation, is different in plants than in animals or yeast. [ABSTRACT FROM AUTHOR]

This work presents new data on human endonuclease VIII-like 2 protein (hNEIL2), a part of DNA glycosylases of the helix–two-turn–helix structural superfamily. While X-ray structure of oNEIL2 (opossum Monodelphis) was resolved partially [1], the structure of hNEIL2 has not yet been determined. This data article describes a powerful combination of hydrogen-deuterium exchange mass spectrometry, homology modeling, and molecular dynamics simulations for protein conformational dynamics analysis.The data supplied in this work are related to the research article entitled “Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry”.