Exchange Mass Spectrometry Experiments Research Articles

Activation of parkin by a molecular glue

Mutations in parkin and PINK1 cause early-onset Parkinson’s disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson’s disease.

Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

The serine phosphorylations in the IRS-1 PIR domain abrogate IRS-1 and IR interaction

Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.

The molecular determinants of classical pathway complement inhibition by OspEF-related proteins of Borrelia burgdorferi

The complement system serves as the first line of defense against invading pathogens by promoting opsonophagocytosis and bacteriolysis. Antibody-dependent activation of complement occurs through the classical pathway and relies on the activity of initiating complement proteases of the C1 complex, C1r and C1s. The causative agent of Lyme disease, Borrelia burgdorferi, expresses two paralogous outer surface lipoproteins of the OspEF-related protein family, ElpB and ElpQ, that act as specific inhibitors of classical pathway activation. We have previously shown that ElpB and ElpQ bind directly to C1r and C1s with high affinity and specifically inhibit C2 and C4 cleavage by C1s. To further understand how these novel protease inhibitors function, we carried out a series of hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments using ElpQ and full-length activated C1s as a model of Elp/protease interaction. Comparison of HDX-MS profiles between unbound ElpQ and the ElpQ/C1s complex revealed a putative C1s-binding site on ElpQ. HDX-MS-guided, site-directed ElpQ mutants were generated and tested for direct binding to C1r and C1s using surface plasmon resonance. Several residues within the C-terminal region of ElpQ were identified as important for protease binding, including a single conserved tyrosine residue that was required for ElpQ- and ElpB-mediated complement inhibition. Collectively, our study identifies key molecular determinants for classical pathway protease recognition by Elp proteins. This investigation improves our understanding of the unique complement inhibitory mechanism employed by Elp proteins which serve as part of a sophisticated complement evasion system present in Lyme disease spirochetes.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Small Molecule Screen Identifies Non-catalytic USP3 Chemical Handle.

Zinc-finger ubiquitin-binding domains (ZnF-UBDs) are noncatalytic domains mostly found in deubiquitylases (DUBs) such as USP3. They represent an underexplored opportunity for the development of deubiquitylase-targeting chimeras (DUBTACs) to pharmacologically induce the deubiquitylation of target proteins. We previously showed that ZnF-UBDs are ligandable domains. Here, a focused small molecule library screen against a panel of 11 ZnF-UBDs led to the identification of compound 59, a ligand engaging the ZnF-UBD of USP3 with a KD of 14 μM. The compound binds the expected C-terminal ubiquitin binding pocket of USP3 as shown by hydrogen-deuterium exchange mass spectrometry experiments and does not inhibit the cleavage of K48-linked diubiquitin by USP3. As such, this molecule is a chemical starting point toward chemical tools that could be used to interrogate the function of the USP3 Znf-UBD and the consequences of recruiting USP3 to ubiquitylated proteins.

Conformational plasticity of RAS Q61 family of neoepitopes results in distinct features for targeted recognition

The conformational landscapes of peptide/human leucocyte antigen (pHLA) protein complexes encompassing tumor neoantigens provide a rationale for target selection towards autologous T cell, vaccine, and antibody-based therapeutic modalities. Here, using complementary biophysical and computational methods, we characterize recurrent RAS55-64 Q61 neoepitopes presented by the common HLA-A*01:01 allotype. We integrate sparse NMR restraints with Rosetta docking to determine the solution structure of NRASQ61K/HLA-A*01:01, which enables modeling of other common RAS55-64 neoepitopes. Hydrogen/deuterium exchange mass spectrometry experiments alongside molecular dynamics simulations reveal differences in solvent accessibility and conformational plasticity across a panel of common Q61 neoepitopes that are relevant for recognition by immunoreceptors. Finally, we predict binding and provide structural models of NRASQ61K antigens spanning the entire HLA allelic landscape, together with in vitro validation for HLA-A*01:191, HLA-B*15:01, and HLA-C*08:02. Our work provides a basis to delineate the solution surface features and immunogenicity of clinically relevant neoepitope/HLA targets for cancer therapy.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture

Abstract Introduction Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS). Methods In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer. Preliminary data The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture. Novel aspect De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

Optimization of hydrogen-deuterium exchange ensemble reweighting (HDXer) to aid the biophysical characterization of proteins.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments provide unique insights into native protein structure and dynamics in solution but have limited spatial resolution. Molecular dynamics (MD) simulations, in contrast, allow for the exploration of protein conformational landscapes with an atomistic level of resolution. However, forcefield inaccuracies, crystallographic biases, and timescale limitations may compromise the validity of the final structural ensemble. Hydrogen Deuterium eXchange Ensemble Reweighting (HDXer) is a maximum entropy approach that was recently developed to (1) quantitatively predict hydrogen-deuterium exchange from a modeled conformational ensemble and (2) refine the candidate ensemble to best fit an experimental HDX-MS dataset. Here, we compare how various computational and experimental parameters impact the performance of HDXer, using extracellular regulated kinase 2 (ERK2) and hen egg white lysozyme (HEWL) as model proteins. Conventional molecular dynamics simulations of ERK2 and HEWL were performed in triplicate using the CHARMM36m, AMBER FF19SB, and Drude forcefields. After predicting the HDX rates of each candidate ensemble, HDXer was used to fit the simulated structures to the corresponding HDX-MS datasets. To assess the influence of simulation length, truncated and strided copies of the full-length simulations were generated. Other factors, including peptide charge state selection, ion mobility mass spectrometry, and independent HDX-MS data acquisitions, were also tested. We find that while some variables (e.g., high vs. low peptide charge states) do not have a significant effect, other parameters (e.g., early vs. late timepoint selection) directly correlate with the disagreement between calculated and experimental deuterium uptake. The results from this benchmarking inform the development of a recommended HDXer workflow and best practices for optimal data acquisition.

The molecular basis for dysfunctional bacterial lipopolysaccharide-mediated immune receptor activation by SARS-CoV-2 spike.

SARS-CoV-2 has caused hundreds of millions of COVID-19 infections worldwide. While COVID-19 presents with various clinical manifestations, severe COVID-19 disease causes dysregulated host immune reactions that trigger onset of sepsis and acute respiratory distress syndrome. Accumulating evidence suggests that lipopolysaccharide (LPS) derived from the outer membranes of Gram-negative bacteria plays an instrumental role in the progression of such inflammatory states. Patients with pre-existing conditions such as diabetes, hypertension, and obesity are at higher risk to develop severe COVID-19 disease and tend to have significantly elevated blood LPS levels. LPS serves as a signal of bacterial infection upon recognition by Toll-like receptor (TLR4) of the innate immune system, but this can also result in over-amplified immune reactions and sepsis. Here, we present the results of molecular simulations and free-energy calculations, supported by in vitro and in vivo assays and hydrogen-deuterium exchange mass spectrometry experiments, which reveal the molecular mechanism by which the envelope spike glycoprotein of SARS-CoV-2 augments hyperinflammation by acting as a conduit in the TLR4 pathway. LPS was found to bind to several conserved pockets on spike across S1 and S2 subunits. S1 affinity for LPS was comparable to that of CD14, a co-receptor used by immune cells to transfer LPS to TLR4. Cell-based assays and reporter mice experiments showed that low concentrations of spike and LPS synergistically induce a strong pro-inflammatory response, thus pinpointing spike's capacity to “boost” innate immune activation. Finally, the loss of a high-affinity binding site in the Omicron spike led to a reduction of its “boosting” capacity, which may translate to the less severe inflammation observed in patients infected with this variant. Collectively, our findings highlight the potential impact of elevated LPS levels and Gram-negative bacterial coinfections in severe COVID-19 complications.

Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

Optimization and Mechanistic Investigations of Novel Allosteric Activators of PKG1α.

Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.

Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry

Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance.

Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen-Deuterium Exchange Mass Spectrometry Experiments.

During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.

Uncovering cryptic pockets in the SARS-CoV-2 spike glycoprotein

The COVID-19 pandemic has prompted a rapid response in vaccine and drug development. Herein, we modeled a complete membrane-embedded SARS-CoV-2 spike glycoprotein and used molecular dynamics simulations with benzene probes designed to enhance discovery of cryptic pockets. This approach recapitulated lipid and host metabolite binding sites previously characterized by cryo-electron microscopy, revealing likely ligand entry routes, and uncovered a novel cryptic pocket with promising druggable properties located underneath the 617–628 loop. A full representation of glycan moieties was essential to accurately describe pocket dynamics. A multi-conformational behavior of the 617–628 loop in simulations was validated using hydrogen-deuterium exchange mass spectrometry experiments, supportive of opening and closing dynamics. The pocket is the site of multiple mutations associated with increased transmissibility found in SARS-CoV-2 variants of concern including Omicron. Collectively, this work highlights the utility of the benzene mapping approach in uncovering potential druggable sites on the surface of SARS-CoV-2 targets.

Structure and Dynamics of Human Perilipin 3 Membrane Association

Lipid droplets (LDs) are a unique organelle that contain an oil core mainly composed of triglycerides that is surrounded by a phospholipid monolayer and coated by LD‐associated proteins called perilipins (PLINs). There are five PLINs in human that generally contain a highly conserved N‐terminal PAT domain of unclear function, followed by variable stretches of amphipathic helices previously implicated in LD association, and a C‐terminal 4‐helix bundle domain. During the process of LD biogenesis, perilipin 3 (PLIN3) is the first PLIN to be recruited to nascent LDs as they emerge from the endoplasmic reticulum. However, how PLIN3 recruitment to LDs is affected by lipid composition, and the structure and dynamics of PLIN membrane association has not been examined in detail. Here, using liposome co‐sedimentation and LD‐flotation assays we found that PLIN3 membrane association was enhanced by membrane packing defects and further promoted by the triglyceride precursors diacylglycerol (DAG) and phosphatidic acid. Hydrogen‐deuterium exchange mass spectrometry experiments identified that both the PAT domain and amphipathic helices undergo a transition upon membrane binding from intrinsically disordered to an ordered state. Using constructs containing different portions of the PAT domain and amphipathic helices, we identify a new functional PAT domain that contains all conserved residues and is strongly recruited to DAG enriched membranes. Consistent with these findings, this newly determined functional PAT domain is predicted to form a triangular helical tertiary structure by alphafold and RoseTTAfold. Taken together, this study reveals molecular details of PLIN3 recruitment to membranes and suggests a role for the PAT domain as a major driver of PLIN3 to DAG enriched membranes, which is consistent with the localization of PLIN3 on early nascent LDs.

Optimizing hydrogen-deuterium exchange ensemble reweighting using candidate ensembles of calmodulin

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments provide unique insights into protein structural dynamics in solution, but this technique is practically restricted to peptide-level resolution. In contrast, molecular dynamics (MD) simulations allow the exploration of a protein’s conformational landscape with atomistic resolution. However, sampling and timescale limitations may mean that the resulting conformations inaccurately reflect the protein’s behavior in solution. A novel maximum entropy reweighing approach, HDXer, was recently developed to (1) predict H/D exchange from an ensemble of generated conformations and (2) reweight individual structures of the modeled ensemble such that the ensemble better conforms with HDX-MS data using a maximum entropy approach.

Moderated Test Statistics to Detect Differential Deuteration in Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

With differential hydrogen/deuterium exchange, differences in the structure and dynamics of protein states can be studied. Detecting statistically significant differentially deuterated peptides is crucial to draw meaningful conclusions about the distinct conformations and dynamics of the protein under study. Here, we introduced a linear model in combination with an empirical Bayes approach to detect differentially deuterated peptides. Using a linear model allows one to test for differences in deuteration between two (two-sample t-test) or more groups (F-statistic), while potentially controlling for the effects of other variables that are not of interest. The empirical Bayes approach improves the estimation of deuteration-level variances, especially in experiments with a low number of replicates. As a consequence, the two sample t-tests and the F-statistic become moderated, resulting in a lower number of false positive and false negative findings. Furthermore, we introduce a thresholded-moderated t-statistic to test if the observed deuteration differences are larger than a specified, biologically relevant difference. Finally, we underline the importance of having a sufficient number of replicates, and the effect of the number of replicates on the power of the statistical significance tests. The R-code for the proposed moderated test statistics is available upon request.