Neuron-specific chromatin disruption at CpG islands and aging-related regions in Kabuki syndrome mice.

Many Mendelian developmental disorders caused by coding variants in epigenetic regulators have now been discovered. Epigenetic regulators are broadly expressed, and each of these disorders typically shows phenotypic manifestations from many different organ systems. An open question is whether the chromatin disruption-the root of the pathogenesis-is similar in the different disease-relevant cell types. This is possible in principle, because all these cell types are subject to effects from the same causative gene, which has the same kind of function (e.g., methylates histones) and is disrupted by the same germline variant. We focus on mouse models for Kabuki syndrome types 1 and 2 and find that the chromatin accessibility changes in neurons are mostly distinct from changes in B or T cells. This is not because the neuronal accessibility changes occur at regulatory elements that are only active in neurons. Neurons, but not B or T cells, show preferential chromatin disruption at CpG islands and at regulatory elements linked to aging. A sensitive analysis reveals that regulatory elements disrupted in B/T cells do show chromatin accessibility changes in neurons, but these are very subtle and of uncertain functional significance. Finally, we are able to identify a small set of regulatory elements disrupted in all three cell types. Our findings reveal the cellular-context-specific effect of variants in epigenetic regulators and suggest that blood-derived episignatures, although useful diagnostically, may not be well suited for understanding the mechanistic basis of neurodevelopment in Mendelian disorders of the epigenetic machinery.

Growth deficiency in a mouse model of Kabuki syndrome 2 bears mechanistic similarities to Kabuki syndrome 1.

Growth deficiency is a characteristic feature of both Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), Mendelian disorders of the epigenetic machinery with similar phenotypes but distinct genetic etiologies. We previously described skeletal growth deficiency in a mouse model of KS1 and further established that a Kmt2d-/- chondrocyte model of KS1 exhibits precocious differentiation. Here we characterized growth deficiency in a mouse model of KS2, Kdm6atm1d/+. We show that Kdm6atm1d/+ mice have decreased femur and tibia length compared to controls and exhibit abnormalities in cortical and trabecular bone structure. Kdm6atm1d/+ growth plates are also shorter, due to decreases in hypertrophic chondrocyte size and hypertrophic zone height. Given these disturbances in the growth plate, we generated Kdm6a-/- chondrogenic cell lines. Similar to our prior in vitro model of KS1, we found that Kdm6a-/- cells undergo premature, enhanced differentiation towards chondrocytes compared to Kdm6a+/+ controls. RNA-seq showed that Kdm6a-/- cells have a distinct transcriptomic profile that indicates dysregulation of cartilage development. Finally, we performed RNA-seq simultaneously on Kmt2d-/-, Kdm6a-/-, and control lines at Days 7 and 14 of differentiation. This revealed surprising resemblance in gene expression between Kmt2d-/- and Kdm6a-/- at both time points and indicates that the similarity in phenotype between KS1 and KS2 also exists at the transcriptional level.

Removing unwanted variation between samples in Hi-C experiments.

Hi-C data are commonly normalized using single sample processing methods, with focus on comparisons between regions within a given contact map. Here, we aim to compare contact maps across different samples. We demonstrate that unwanted variation, of likely technical origin, is present in Hi-C data with replicates from different individuals, and that properties of this unwanted variation change across the contact map. We present band-wise normalization and batch correction, a method for normalization and batch correction of Hi-C data and show that it substantially improves comparisons across samples, including in a quantitative trait loci analysis as well as differential enrichment across cell types.

Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Precision pharmacological reversal of strain-specific diet-induced metabolic syndrome in mice informed by epigenetic and transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.

Quantification and statistical modeling of droplet-based single-nucleus RNA-sequencing data.

In complex tissues containing cells that are difficult to dissociate, single-nucleus RNA-sequencing (snRNA-seq) has become the preferred experimental technology over single-cell RNA-sequencing (scRNA-seq) to measure gene expression. To accurately model these data in downstream analyses, previous work has shown that droplet-based scRNA-seq data are not zero-inflated, but whether droplet-based snRNA-seq data follow the same probability distributions has not been systematically evaluated. Using pseudonegative control data from nuclei in mouse cortex sequenced with the 10x Genomics Chromium system and mouse kidney sequenced with the DropSeq system, we found that droplet-based snRNA-seq data follow a negative binomial distribution, suggesting that parametric statistical models applied to scRNA-seq are transferable to snRNA-seq. Furthermore, we found that the quantification choices in adapting quantification mapping strategies from scRNA-seq to snRNA-seq can play a significant role in downstream analyses and biological interpretation. In particular, reference transcriptomes that do not include intronic regions result in significantly smaller library sizes and incongruous cell type classifications. We also confirmed the presence of a gene length bias in snRNA-seq data, which we show is present in both exonic and intronic reads, and investigate potential causes for the bias.

Direct coordination of pterin to FeII enables neurotransmitter biosynthesis in the pterin-dependent hydroxylases.

The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes activate oxygen using a pterin cofactor and an aromatic amino acid substrate bound to the FeII active site to form a highly reactive FeIV = O species that initiates substrate oxidation. In this study, using tryptophan hydroxylase, we have kinetically generated a pre-FeIV = O intermediate and characterized its structure as a FeII-peroxy-pterin species using absorption, Mössbauer, resonance Raman, and nuclear resonance vibrational spectroscopies. From parallel characterization of the pterin cofactor and tryptophan substrate-bound ternary FeII active site before the O2 reaction (including magnetic circular dichroism spectroscopy), these studies both experimentally define the mechanism of FeIV = O formation and demonstrate that the carbonyl functional group on the pterin is directly coordinated to the FeII site in both the ternary complex and the peroxo intermediate. Reaction coordinate calculations predict a 14 kcal/mol reduction in the oxygen activation barrier due to the direct binding of the pterin carbonyl to the FeII site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes.

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.

Promoter CpG Density Predicts Downstream Gene Loss-of-Function Intolerance.

The aggregation and joint analysis of large numbers of exome sequences has recently made it possible to derive estimates of intolerance to loss-of-function (LoF) variation for human genes. Here, we demonstrate strong and widespread coupling between genic LoF intolerance and promoter CpG density across the human genome. Genes downstream of the most CpG-rich promoters (top 10% CpG density) have a 67.2% probability of being highly LoF intolerant, using the LOEUF metric from gnomAD. This is in contrast to 7.4% of genes downstream of the most CpG-poor (bottom 10% CpG density) promoters. Combining promoter CpG density with exonic and promoter conservation explains 33.4% of the variation in LOEUF, and the contribution of CpG density exceeds the individual contributions of exonic and promoter conservation. We leverage this to train a simple and easily interpretable predictive model that outperforms other existing predictors and allows us to classify 1,760 genes-which are currently unascertained in gnomAD-as highly LoF intolerant or not. These predictions have the potential to aid in the interpretation of novel variants in the clinical setting. Moreover, our results reveal that high CpG density is not merely a generic feature of human promoters but is preferentially encountered at the promoters of the most selectively constrained genes, calling into question the prevailing view that CpG islands are not subject to selection.

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing.

Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.

Addressing the mean-correlation relationship in co-expression analysis.

Estimates of correlation between pairs of genes in co-expression analysis are commonly used to construct networks among genes using gene expression data. As previously noted, the distribution of such correlations depends on the observed expression level of the involved genes, which we refer to this as a mean-correlation relationship in RNA-seq data, both bulk and single-cell. This dependence introduces an unwanted technical bias in co-expression analysis whereby highly expressed genes are more likely to be highly correlated. Such a relationship is not observed in protein-protein interaction data, suggesting that it is not reflecting biology. Ignoring this bias can lead to missing potentially biologically relevant pairs of genes that are lowly expressed, such as transcription factors. To address this problem, we introduce spatial quantile normalization (SpQN), a method for normalizing local distributions in a correlation matrix. We show that spatial quantile normalization removes the mean-correlation relationship and corrects the expression bias in network reconstruction.

Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis.

DNA methylation is tightly regulated throughout mammalian development, and altered DNA methylation patterns are a general hallmark of cancer. The methylcytosine dioxygenase TET2 is frequently mutated in hematological disorders, including acute myeloid leukemia (AML), and has been suggested to protect CG dinucleotide (CpG) islands and promoters from aberrant DNA methylation. In this study, we present a novel Tet2-dependent leukemia mouse model that closely recapitulates gene expression profiles and hallmarks of human AML1-ETO-induced AML. Using this model, we show that the primary effect of Tet2 loss in preleukemic hematopoietic cells is progressive and widespread DNA hypermethylation affecting up to 25% of active enhancer elements. In contrast, CpG island and promoter methylation does not change in a Tet2-dependent manner but increases relative to population doublings. We confirmed this specific enhancer hypermethylation phenotype in human AML patients with TET2 mutations. Analysis of immediate gene expression changes reveals rapid deregulation of a large number of genes implicated in tumorigenesis, including many down-regulated tumor suppressor genes. Hence, we propose that TET2 prevents leukemic transformation by protecting enhancers from aberrant DNA methylation and that it is the combined silencing of several tumor suppressor genes in TET2 mutated hematopoietic cells that contributes to increased stem cell proliferation and leukemogenesis.

A metabolomics approach identified toxins associated with uremic symptoms in advanced chronic kidney disease.

Uremic symptoms are common in patients with advanced chronic kidney disease, but the toxins that cause these symptoms are unknown. To evaluate this, we performed a cross-sectional study of the 12 month post-randomization follow-up visit of Modification of Diet in Renal Disease (MDRD) participants reporting uremic symptoms who also had available stored serum. We quantified 1,163 metabolites by liquid chromatography-tandem mass spectrometry. For each uremic symptom, we calculated a score as the severity multiplied by the number of days the symptom was experienced. We analyzed the associations of the individual symptom scores with metabolites using linear models with empirical Bayesian inference, adjusted for multiple comparisons. Among 695 participants, the mean measured glomerular filtration rate (mGFR) was 28 mL/min/1.73 m2. Uremic symptoms were more common in the subgroup of 214 patients with an mGFR under 20 mL/min/1.73 m2 (mGFR under 20 subgroup) than in the full group. For all metabolites with significant associations, the direction of the association was concordant in the full group and the subgroup. For gastrointestinal symptoms (bad taste, loss of appetite, nausea, and vomiting), eleven metabolites were associated with symptoms. For neurologic symptoms (decreased alertness, falling asleep during the day, forgetfulness, lack of pep and energy, and tiring easily/weakness), seven metabolites were associated with symptoms. Associations were consistent across sensitivity analyses. Thus, our proof-of-principle study demonstrates the potential for metabolomics to understand metabolic pathways associated with uremic symptoms. Larger, prospective studies with external validation are needed.

Coexpression patterns define epigenetic regulators associated with neurological dysfunction.

Coding variants in epigenetic regulators are emerging as causes of neurological dysfunction and cancer. However, a comprehensive effort to identify disease candidates within the human epigenetic machinery (EM) has not been performed; it is unclear whether features exist that distinguish between variation-intolerant and variation-tolerant EM genes, and between EM genes associated with neurological dysfunction versus cancer. Here, we rigorously define 295 genes with a direct role in epigenetic regulation (writers, erasers, remodelers, readers). Systematic exploration of these genes reveals that although individual enzymatic functions are always mutually exclusive, readers often also exhibit enzymatic activity (dual-function EM genes). We find that the majority of EM genes are very intolerant to loss-of-function variation, even when compared to the dosage sensitive transcription factors, and we identify 102 novel EM disease candidates. We show that this variation intolerance is driven by the protein domains encoding the epigenetic function, suggesting that disease is caused by a perturbed chromatin state. We then describe a large subset of EM genes that are coexpressed within multiple tissues. This subset is almost exclusively populated by extremely variation-intolerant genes and shows enrichment for dual-function EM genes. It is also highly enriched for genes associated with neurological dysfunction, even when accounting for dosage sensitivity, but not for cancer-associated EM genes. Finally, we show that regulatory regions near epigenetic regulators are genetically important for common neurological traits. These findings prioritize novel disease candidate EM genes and suggest that this coexpression plays a functional role in normal neurological homeostasis.

TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells.

The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.

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.

PNGase H + variant from Rudaea cellulosilytica with improved deglycosylation efficiency for rapid analysis of eukaryotic N-glycans and hydrogen deuterium exchange mass spectrometry analysis of glycoproteins.

The analysis of glycoproteins and the comparison of protein N-glycosylation from different eukaryotic origins require unbiased and robust analytical workflows. The structural and functional analysis of vertebrate protein N-glycosylation currently depends extensively on bacterial peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidases (PNGases), which are indispensable enzymatic tools in releasing asparagine-linked oligosaccharides (N-glycans) from glycoproteins. So far, only limited PNGase candidates are available for N-glycans analysis, and particularly the analysis of plant and invertebrate N-glycans is hampered by the lack of suitable PNGases. Furthermore, liquid chromatography-mass spectrometry (LC-MS) workflows, such as hydrogen deuterium exchange mass spectrometry (HDX-MS), require a highly efficient enzymatic release of N-glycans at low pH values to facilitate the comprehensive structural analysis of glycoproteins. Herein, we describe a previously unstudied superacidic bacterial N-glycanase (PNGase H+ ) originating from the soil bacterium Rudaea cellulosilytica (Rc), which has significantly improved enzymatic properties compared to previously described PNGase H+ variants. Active and soluble recombinant PNGase Rc was expressed at a higher protein level (3.8-fold) and with higher specific activity (~56% increase) compared to the currently used PNGase H+ variant from Dyella japonicum (Dj). Recombinant PNGase Rc was able to deglycosylate the glycoproteins horseradish peroxidase and bovine lactoferrin significantly faster than PNGase Dj (10 min vs. 6 h). The versatility of PNGase Rc was demonstrated by releasing N-glycans from a diverse array of samples such as peach fruit, king trumpet mushroom, mouse serum, and the soil nematode Caenorhabditis elegans. The presence of only two disulfide bonds shown in the AlphaFold protein model (so far all other superacidic PNGases possess more disulfide bonds) could be corroborated by intact mass- and peptide mapping analysis and provides a possible explanation for the improved recombinant expression yield of PNGase Rc.

The expression of HSP70 in skeletal muscle is not associated with glycogen availability during recovery following prolonged exercise in elite endurance athletes.

The 70-kDa heat shock protein (HSP70) is a ubiquitous molecular chaperone which is highly inducible by cellular stress such as exercise. To investigate the role of muscle glycogen content on the HSP70 expression, muscle glycogen was manipulated by consumption of either water (H2O) or a carbohydrate-enriched diet (CHO) during recovery from 4 h of glycogen-depleting cycling exercise in fourteen elite endurance athletes. Muscle biopsies were obtained pre- and post-exercise, and after 4 and 24 h of recovery, and analyzed for HSP70 mRNA expression, as well as HSP70 protein expression and muscle glycogen within the same skeletal muscle fibers using immunohistochemistry. Exercise reduced glycogen by 59 ± 10% (P < 0.0001). After 4 h of recovery, glycogen approached resting levels in the CHO group (86% of pre, P = 0.28) but remained suppressed in the H2O group (41% of pre, P < 0.001) (group × time interaction: P = 0.002). Importantly, both the HSP70 mRNA (+ 1.6-fold (+ 0.28/- 0.24), P = 0.02) and protein expression (+ 147 ± 99%, P < 0.0001) was substantially increased after exercise and remained elevated in both groups after 4 h of recovery, despite clear differences in muscle glycogen content. Thus, muscle glycogen content was not related to the variation in single fiber HSP70 expression at the 4-h time-point (r2 = 0.004). In conclusion, muscle HSP70 expression remained elevated during recovery from prolonged exercise in highly trained skeletal muscle, irrespective of muscle glycogen availability.

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.