Inverse effects of APOC2 and ANGPTL4 on the conformational dynamics of lid-anchoring structures in lipoprotein lipase.

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 LPL's α/ß-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 APOC2's C-terminal α-helix binds to regions of LPL surrounding the catalytic pocket. Remarkably, APOC2's 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 LPL's active site.

The intrinsic instability of the hydrolase domain of lipoprotein lipase facilitates its inactivation by ANGPTL4-catalyzed unfolding.

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/ß-hydrolase domain. Here, we show that this unfolding is initiated by the binding of ANGPTL4 to sequences near LPL's catalytic site, including ß2, ß3-α3, and the lid. Using pulse-labeling hydrogen‒deuterium exchange mass spectrometry, we found that ANGPTL4 binding initiates conformational changes that are nucleated on ß3-α3 and progress to ß5 and ß4-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/ß-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.

Effect of a Lactococcus lactis culture supernatant on diarrhea and performance parameters of piglets in the post-weaning period and on expression of the faeG gene in vitro.

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+. Animals and procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL. Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed (P < 0.0001) relative to that in the untreated control. Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259.

Effet d'un surnageant de culture de Lactococcus lactis sur la diarrhée et les paramètres de performance des porcelets en période post-sevrage et sur l'expression du gène faeG in vitro. Objectifs: Évaluer les effets d'un surnageant acellulaire de Lactococcus lactis (CFSM) sur les paramètres de performance et de diarrhée et la présence d'E. coli entérotoxinogène F4+ (ETEC) chez les porcelets en post-sevrage, et évaluer l'effet in vitro du CFSM sur l'expression du gène faeG dans un E. coli F4+. Animaux et procédure: Dans 3 essais portant sur 90 porcelets par essai, les porcs ont reçu un placebo ou 1 des 2 traitements CFSM et ont été observés pour détecter la diarrhée et leurs performances. Des prélèvements fécaux ont été effectués pour déterminer la présence d'ETEC. La RT-PCR quantitative a été utilisée pour évaluer l'expression du gène faeG dans E. coli 21259 après traitement avec CFSM à 50 mg/mL. Résultats: Le CFSM administré pendant 14 jours à une dose de 24 mg/kg de poids corporel (2X) a réduit les paramètres liés à la diarrhée par rapport au placebo. La RT-PCR quantitative a montré que, chez E. coli 21259 traité avec CFSM à 50 mg/mL, l'expression du gène faeG était significativement réprimée (P < 0,0001) par rapport à celle du témoin non traité. Conclusion: Le CFSM évalué a réduit la fréquence et la prévalence de la diarrhée sur le terrain. Le traitement in vitro a eu un effet inhibiteur sur l'expression du gène faeG chez F4+ E. coli 21259.(Traduit par Dr Serge Messier).

Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties.

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism.

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.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Lipid Peroxidation Products HNE and ONE Promote and Stabilize Alpha-Synuclein Oligomers by Chemical Modifications.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

Probing the Conformational Dynamics of Affinity-Enhanced T Cell Receptor Variants upon Binding the Peptide-Bound Major Histocompatibility Complex by Hydrogen/Deuterium Exchange Mass Spectrometry.

Binding of the T cell receptor (TCR) to its cognate, peptide antigen-loaded major histocompatibility complex (pMHC) is a key interaction for triggering T cell activation and ultimately elimination of the target cell. Despite the importance of this interaction for cellular immunity, a comprehensive molecular understanding of TCR specificity and affinity is lacking. We conducted hydrogen/deuterium exchange mass spectrometry (HDX-MS) analyses of individual affinity-enhanced TCR variants and clinically relevant pMHC class I molecules (HLA-A*0201/NY-ESO-1157-165) to investigate the causality between increased binding affinity and conformational dynamics in TCR-pMHC complexes. Differential HDX-MS analyses of TCR variants revealed that mutations for affinity enhancement in TCR CDRs altered the conformational response of TCR to pMHC ligation. Improved pMHC binding affinity was in general observed to correlate with greater differences in HDX upon pMHC binding in modified TCR CDR loops, thereby providing new insights into the TCR-pMHC interaction. Furthermore, a specific point mutation in the ß-CDR3 loop of the NY-ESO-1 TCR associated with a substantial increase in binding affinity resulted in a substantial change in pMHC binding kinetics (i.e., very slow kon, revealed by the detection of EX1 HDX kinetics), thus providing experimental evidence for a slow induced-fit binding mode. We also examined the conformational impact of pMHC binding on an unrelated TRAV12-2 gene-encoded TCR directed against the immunodominant MART-126-35 cancer antigen restricted by HLA-A*0201. Our findings provide a molecular basis for the observed TRAV12-2 gene bias in natural CD8+ T cell-based immune responses against the MART-1 antigen, with potential implications for general ligand discrimination and TCR cross-reactivity processes.

Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling.

Ultraviolet photodissociation (UVPD) has recently been introduced as an ion activation method for the determination of single-residue deuterium levels in H/D exchange tandem mass spectrometry experiments. In this regard, it is crucial to know which fragment ion types can be utilized for this purpose. UVPD yields rich product ion spectra where all possible backbone fragment ion types (a/x, b/y, and c/z) are typically observed. Here we provide a detailed investigation of the level of H/D scrambling for all fragment ion types upon UVPD of the peptide scrambling probe P1 (HHHHHHIIKIIK) using an Orbitrap tribrid mass spectrometer equipped with a solid-state 213 nm UV laser. The most abundant UVPD-generated fragment ions (i.e., b/y ions) exhibit extensive H/D scrambling. Similarly, a/x and c/z ions have also undergone H/D scrambling due to UV-induced heating of the precursor ion population. Therefore, dominant b/y ions upon UVPD of protonated peptides are a strong indicator for the occurrence of extensive H/D scrambling of the precursor ion population. In contrast to peptide P1, UV-irradiation of ubiquitin did not induce H/D scrambling in the nonfragmented precursor ion population. However, the UVPD-generated b2 and a4 ions from ubiquitin exhibit extensive H/D scrambling. To minimize H/D scrambling, short UV-irradiation time and high gas pressures are recommended.

A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase.

The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (kon) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.

Characterization of the TyrR Regulon in the Rhizobacterium Enterobacter ludwigii UW5 Reveals Overlap with the CpxR Envelope Stress Response.

The TyrR transcription factor controls the expression of genes for the uptake and biosynthesis of aromatic amino acids in Escherichia coli In the plant-associated and clinically significant proteobacterium Enterobacter ludwigii UW5, the TyrR orthologue was previously shown to regulate genes that encode enzymes for synthesis of the plant hormone indole-3-acetic acid and for gluconeogenesis, indicating a broader function for the transcription factor. This study aimed to delineate the TyrR regulon of E. ludwigii by comparing the transcriptomes of the wild type and a tyrR deletion strain. In E. ludwigii, TyrR positively or negatively regulates the expression of over 150 genes. TyrR downregulated expression of envelope stress response regulators CpxR and CpxP through interaction with a DNA binding site in the intergenic region between divergently transcribed cpxP and cpxR Repression of cpxP was alleviated by tyrosine. Methyltransferase gene dmpM, which is possibly involved in antibiotic synthesis, was strongly activated in the presence of tyrosine and phenylalanine by TyrR binding to its promoter region. TyrR also regulated expression of genes for aromatic catabolism and anaerobic respiration. Our findings suggest that the E. ludwigii TyrR regulon has diverged from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrate the expansion and plasticity of transcription factor regulons.IMPORTANCE Genome-wide RNA sequencing revealed a broader regulatory role for the TyrR transcription factor in the ecologically versatile bacterium Enterobacter ludwigii beyond that of aromatic amino acid synthesis and transport that constitute the role of the TyrR regulon of E. coli In E. ludwigii, a plant symbiont and human gut commensal, the TyrR regulon is expanded to include genes that are beneficial for plant interactions and response to stresses. Identification of the genes regulated by TyrR provides insight into the mechanisms by which the bacterium adapts to its environment.

Did evolution create a flexible ligand-binding cavity in the urokinase receptor through deletion of a plesiotypic disulfide bond?

The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7-8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7-8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences.

Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) enables the study of protein dynamics by measuring the time-resolved deuterium incorporation into a protein incubated in D2O. Using electron-based fragmentation in the gas phase it is possible to measure deuterium uptake at single-residue resolution. However, a prerequisite for this approach is that the solution-phase labeling is conserved in the gas phase prior to precursor fragmentation. It is therefore essential to reduce or even avoid intramolecular hydrogen/deuterium migration, which causes randomization of the deuterium labels along the peptide (hydrogen scrambling). Here, we describe an optimization strategy for reducing scrambling to a negligible level while minimizing the impact on sensitivity on a high-resolution Q-TOF equipped with ETD and an electrospray ionization interface consisting of a glass transfer capillary followed by a dual ion funnel. In our strategy we narrowed down the optimization to two accelerating potentials, and we defined the optimization of these in a simple rule by accounting for their interdependency in relation to scrambling and transmission efficiency. Using this rule, we were able to reduce scrambling from 75% to below 5% on average using the highly scrambling-sensitive quadruply charged P1 peptide scrambling probe resulting in a minor 33% transmission loss. To demonstrate the applicability of this approach, we probe the dynamics of certain regions in cytochrome c.

Rapid morphological evolution in placental mammals post-dates the origin of the crown group.

Resolving the timing and pattern of early placental mammal evolution has been confounded by conflict among divergence date estimates from interpretation of the fossil record and from molecular-clock dating studies. Despite both fossil occurrences and molecular sequences favouring a Cretaceous origin for Placentalia, no unambiguous Cretaceous placental mammal has been discovered. Investigating the differing patterns of evolution in morphological and molecular data reveals a possible explanation for this conflict. Here, we quantified the relationship between morphological and molecular rates of evolution. We show that, independent of divergence dates, morphological rates of evolution were slow relative to molecular evolution during the initial divergence of Placentalia, but substantially increased during the origination of the extant orders. The rapid radiation of placentals into a highly morphologically disparate Cenozoic fauna is thus not associated with the origin of Placentalia, but post-dates superordinal origins. These findings predict that early members of major placental groups may not be easily distinguishable from one another or from stem eutherians on the basis of skeleto-dental morphology. This result supports a Late Cretaceous origin of crown placentals with an ordinal-level adaptive radiation in the early Paleocene, with the high relative rate permitting rapid anatomical change without requiring unreasonably fast molecular evolutionary rates. The lack of definitive Cretaceous placental mammals may be a result of morphological similarity among stem and early crown eutherians, providing an avenue for reconciling the fossil record with molecular divergence estimates for Placentalia.

The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed ß2-Microglobulin through Distinct Binding Sites.

T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR-pMHC complex formation significantly stabilizes distinct CDR loops of the TCR, it does not trigger structural changes in receptor segments remote from the binding interface. Intriguingly, our HDX measurements reveal that the TCR α-constant domain (C- and F-strand) directly interacts with the unbound MHC light chain, ß2-microglobulin (ß2m). Surface plasmon resonance measurements corroborated a binding event between TCR and ß2m with a dissociation constant of 167 ± 20 µM. We propose a model structure for the TCR-ß2m complex based on a refined protein-protein docking approach driven by HDX data and information from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of ß2m on T-cell cytotoxicity, suggesting an alternate role for ß2m binding. Overall, we show that binding of ß2m to the TCR occurs in vitro and, as such, not only should be considered in structure-function studies of the TCR-pMHC complex but also could play a hitherto unidentified role in T-cell function in vivo.

Epitope-dependent Functional Effects of Celiac Disease Autoantibodies on Transglutaminase 2.

Transglutaminase 2 (TG2) is a Ca2+-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2. Binding of the mAbs induced allosteric changes in TG2. Thus, we aimed to determine whether these mAbs could influence enzymatic activity through modulation of TG2 susceptibility to oxidative inactivation and Ca2+ affinity. All tested epitope 1 mAbs, as well as 679-14-D04, which recognizes a previously uncharacterized epitope, prevented oxidative inactivation and increased Ca2+ sensitivity of TG2. We have identified crucial residues for binding of 679-14-D04 located within a Ca2+ binding site. Epitope 1 mAbs and 679-14-D04, although recognizing separate epitopes, behaved similarly when assessing their effect on TG2 conformation, suggesting that the shared effects on TG2 function can be explained by induction of the same conformational changes. None of the mAbs targeting other epitopes showed these effects, but epitope 2 mAbs reduced the rate of TG2-catalyzed reactions. Collectively, these effects could be relevant to the pathogenesis of CD. In A20 B cells transduced with TG2-specific B-cell receptor, epitope 2-expressing cells had poorer uptake of TG2-gluten complexes and were less efficient in gluten epitope presentation to T cells than cells expressing an epitope 1 receptor. Thus, the ability of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients.

An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a ß4A-ß5A ß-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.

Uranyl Photocleavage of Phosphopeptides Yields Truncated C-Terminally Amidated Peptide Products.

The uranyl ion (UO22+ ) binds phosphopeptides with high affinity, and when irradiated with UV-light, it can cleave the peptide backbone. In this study, high-accuracy tandem mass spectrometry and enzymatic assays were used to characterise the photocleavage products resulting from the uranyl photocleavage reaction of a tetraphosphorylated ß-casein model peptide. We show that the primary photocleavage products of the uranyl-catalysed reaction are C-terminally amidated. This could be of great interest to the pharmaceutical industry, as efficient peptide amidation reactions are one of the top challenges in green pharmaceutical chemistry.

Dissecting the interaction between transglutaminase 2 and fibronectin.

In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell-matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN.