Hydrogen-deuterium Exchange Experiments Research Articles

CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.

Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

JNJ-7184, a respiratory syncytial virus inhibitor targeting the connector domain of the viral polymerase

Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.

Formulation development of a stable influenza recombinant neuraminidase vaccine candidate

ABSTRACT Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a “closed” to an “open” conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.

Structural Basis for p19 Targeting by Anti–IL-23 Biologics: Correlations with Short- and Long-Term Efficacy in Psoriasis

IL-23 is central to psoriasis pathogenesis. Biologics targeting IL-23 are important therapies against psoriasis. IL-23 inhibitors risankizumab, tildrakizumab, and guselkumab bind the IL-23 p19 subunit, whereas ustekinumab binds p40; however, the structural composition of the IL-23-binding epitopes and how these molecular properties relate to clinical efficacy are not known. Utilizing epitope data derived from hydrogen-deuterium exchange or crystallographic experiments, we mapped inhibitor epitope locations, hydrophobicity, and surface charge onto the IL-23 surface. Molecular properties of each inhibitor epitope, including solvent-accessible surface area, were correlated to binding affinity, kinetic values, and clinical efficacy scores for plaque psoriasis through linear regression analysis. Each IL-23 inhibitor binds an epitope with a unique size, composition, and location except for a 10-residue overlap region outside of the IL-23 receptor epitope. We observed strong correlations between epitope surface area and KD and koff but not kon. Epitope surface area, KD, and koff were further associated with short-term (10-16 weeks) and long-term (44-60 weeks) clinical efficacy according to PASI-90 responses, with risankizumab demonstrating highest efficacy among IL-23 biologics. In contrast, kon, epitope hydrophobicity, polarity, and charge content did not correlate with efficacy. These data exemplify how molecular principles of medications within a therapeutic class can explain their differential clinical responses.

G protein-coupled receptor pharmacology - insights from mass spectrometry.

G protein-coupled receptors (GPCRs) are key drug targets due to their involvement in many physiological processes. The complexity of receptor pharmacology however is influenced by multiple interactions with various types of ligands and protein transducers representing significant challenges for drug discovery. The ability of mass spectrometry to observe both the binding of ligand molecules such as lipids, ions or drugs and their impact on interaction with transducers provides an exciting opportunity to probe many aspects that are difficult to track directly in cell-based systems. From the early days, when hydrogen deuterium exchange (HDX) experiments were used to probe the different conformations of GPCRs, through to the most recent insights in which the intact receptor-G protein/arrestin complexes associated with small molecules can be preserved by mass spectrometry, this review highlights the potential of mass spectrometry techniques for in-depth investigations of GPCR biology. Herein, we will describe the utility of mass spectrometry (MS) including HDX-MS, cross-linking and native-MS, in investigating GPCR pharmacology. Specifically, we will include ligand/drug interactions and Gi/s protein-coupling and illustrate and how these techniques can lead to the discovery of endogenous allosteric ligands and thereby offer a new perspective for drug discovery of GPCRs. Significance Statement GPCRs represent the largest family of druggable protein targets that interact with a diverse array of ligands, from metal ions and small molecules through to peptides and proteins. These extracellular stimuli are translated allosterically into intracellular signals mediated by transducer proteins. Typically, activation of GPCRs relies on binding of endogenous or exogenous agonists to stabilize the receptors in active conformations. Recent developments in MS enable capture of the extent of GPCR activation and discovery of allosteric modulators of downstream signaling.

Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.

Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis.

Antifreeze glycoprotein (AFGP), which inhibits the freezing of water, is highly O-glycosylated with a disaccharide, d-Galβ1-3-d-GalNAcα (GalGalNAc). To elucidate the function of the sugar residues for antifreeze activity at the molecular level, we conducted a total chemical synthesis of partially sugar deleted AFGP derivatives, and unnatural forms of AFGPs incorporating glucose (Glc)-type sugars instead of galactose (Gal)-type sugars. These elaborated AFGP derivatives demonstrated that the stereochemistry of each sugar residue on AFGPs precisely correlates with the antifreeze activity. A hydrogen-deuterium exchange experiment using synthetic AFGPs revealed a different dynamic behavior of water around sugar residues depending on the sugar structures. These results indicate that sugar residues on AFGP form a unique dynamic water phase that disturbs the absorbance of water molecules onto the ice surface, thereby inhibiting freezing.

Artificial intelligence-based HDX (AI-HDX) prediction reveals fundamental characteristics to protein dynamics: Mechanisms on SARS-CoV-2 immune escape

Three-dimensional structure and dynamics are essential for protein function. Advancements in hydrogen-deuterium exchange (HDX) techniques enable probing protein dynamic information in physiologically relevant conditions. HDX-coupled mass spectrometry (HDX-MS) has been broadly applied in pharmaceutical industries. However, it is challenging to obtain dynamics information at the single amino acid resolution and time consuming to perform the experiments and process the data. Here, we demonstrate the first deep learning model, artificial intelligence-based HDX (AI-HDX), that predicts intrinsic protein dynamics based on the protein sequence. It uncovers the protein structural dynamics by combining deep learning, experimental HDX, sequence alignment, and protein structure prediction. AI-HDX can be broadly applied to drug discovery, protein engineering, and biomedical studies. As a demonstration, we elucidated receptor-binding domain structural dynamics as a potential mechanism of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody efficacy and immune escape. AI-HDX fundamentally differs from the current AI tools for protein analysis and may transform protein design for various applications.

Apparatus for Automated Continuous Hydrogen Deuterium Exchange Mass Spectrometry Measurements from Milliseconds to Hours.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the "static" picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4-5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of "off-the-shelf" LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.

Quaternary glucocorticoid receptor structure highlights allosteric interdomain communication.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that binds DNA and assembles co-regulator complexes to regulate gene transcription. GR agonists are widely prescribed to people with inflammatory and autoimmune diseases. Here we present high-resolution, multidomain structures of GR in complex with ligand, DNA and co-regulator peptide. The structures reveal how the receptor forms an asymmetric dimer on the DNA and provide a detailed view of the domain interactions within and across the two monomers. Hydrogen-deuterium exchange and DNA-binding experiments demonstrate that ligand-dependent structural changes are communicated across the different domains in the full-length receptor. This study demonstrates how GR forms a distinct architecture on DNA and how signal transmission can be modulated by the ligand pharmacophore, provides a platform to build a new level of understanding of how receptor modifications can drive disease progression and offers key insight for future drug design.

PrIntMap-R: An Online Application for Intraprotein Intensity and Peptide Visualization from Bottom-Up Proteomics.

Bottom-up proteomics (BUP) produces rich data, but visualization and analysis are time-consuming and often require programming skills. Many tools analyze these data at the proteome-level, but fewer options exist for individual proteins. Sequence coverage maps are common, but do not proportion peptide intensity. Abundance-based visualization of sequence coverage facilitates detection of protein isoforms, domains, potential truncation sites, peptide "hot-spots", and localization of post-translational modifications (PTMs). Redundant stacked-sequence coverage is an important tool in designing hydrogen-deuterium exchange (HDX) experiments. Visualization tools often lack graphical and tabular-export of processed data which complicates publication of results. Quantitative peptide abundance across amino acid sequences is an essential and missing tool in proteomics toolkits. Here we created PrIntMap-R, an online application that only requires peptide files from a database search and FASTA protein sequences. PrIntMap-R produces a variety of plots for quantitative visualization of coverage; annotation of specific sequences, PTM's, and comparisons of one or many samples overlaid with calculated fold-change or several intensity metrics. We show use-cases including protein phosphorylation, identification of glycosylation, and the optimization of digestion conditions for HDX experiments. PrIntMap-R is freely available, open source, and can run online with no installation, or locally by downloading source code from GitHub.

Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen-deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face π-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a molecular staple with Phe37 that stabilizes a nonfunctional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations, namely, that they must avoid inappropriately stabilizing nonfunctional protein conformations.

Validation of the solution structure of dimerization domain of PRC1.

Cell-cycle dependent proteins are indispensible for the accurate division of cells, a group of proteins called Microtubule-associated proteins (MAPs) are important to cell division as it bind microtubules and participate with other co-factors to form the spindle midbody, which works as the workhorse of cell-division. PRC1 is a distinguishing member of MAPs, as it is a human MAP and works as the key in mediating daughter cell segregation in ana-phase and telo-phase. The physiological significance of PRC1 calls for a high resolution three-dimensional structure. The crystal structure of PRC1 was published but has low resolution (>3 Å) and incomplete sidechains, placing hurdles to understanding the structure-function relationships of PRC1, therefore, we determined the high-resolution solution structure of PRC1’s dimerization domain using NMR spectroscopy. Significant differences between the crystal structure and the solution structure can be observed, the main differences center around the N terminus and the end of the alpha-Helix H2. Furthermore, detailed structure analyses revealed that the hydrophobic core packing of the solution and crystal structures are also different. To validate the solution structure, we used Hydrogen-deuterium exchange experiments that address the structural discrepancies between the crystal and solution structure; we also generated mutants that are key to the differences in the crystal and solution structures, measuring its structural or thermal stability by NMR spectroscopy and Fluorescence Thermal Shift Assays. These results suggest that N terminal residues are key to the integrity of the whole protein, and the solution structure of the dimerization domain better reflects the conformation PRC1 adopted in solution conditions.

1H, 13C and 15N resonance assignments of the first BIR domain of cellular inhibitor of apoptosis protein 1.

Cellular inhibitor of apoptosis protein-1 (cIAP-1) is member of inhibitor of apoptosis proteins (IAPs) which can affect apoptosis through interactions with caspases. cIAP-1 is a multi-domain protein and able to regulate apoptosis through interactions with proteins such as caspases and possesses E3 ligase activity. Human cIAP-1 contains three baculovirus IAP repeat (BIR) domains which are critical for protein-protein interactions. Here, we report NMR resonance assignments of the first BIR domain of human cIAP. Its secondary structures in solution were determined based on the assigned resonances. The dynamics of this domain was obtained, and our hydrogen-deuterium exchange experiment reveals that the first helix in BIR1 is exposed to the solvent. The availability of assignments of backbone and side chain resonances will be useful for probing protein-protein interactions.

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the β-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

Interpreting Hydrogen-Deuterium Exchange Experiments with Molecular Simulations: Tutorials and Applications of the HDXer Ensemble Reweighting Software [Article v1.0

Hydrogen-deuterium exchange (HDX) is a comprehensive yet detailed probe of protein structure and dynamics and, coupled to mass spectrometry, has become a powerful tool for investigating an increasingly large array of systems. Computer simulations are often used to help rationalize experimental observations of exchange, but interpretations have frequently been limited to simple, subjective correlations between microscopic dynamical fluctuations and the observed macroscopic exchange behavior. With this in mind, we previously developed the HDX ensemble reweighting approach and associated software, HDXer, to aid the objective interpretation of HDX data using molecular simulations. HDXer has two main functions; first, to compute H-D exchange rates that describe each structure in a candidate ensemble of protein structures, for example from molecular simulations, and second, to objectively reweight the conformational populations present in a candidate ensemble to conform to experimental exchange data. In this article, we first describe the HDXer approach, theory, and implementation. We then guide users through a suite of tutorials that demonstrate the practical aspects of preparing experimental data, computing HDX levels from molecular simulations, and performing ensemble reweighting analyses. Finally we provide a practical discussion of the capabilities and limitations of the HDXer methods including recommendations for a user's own analyses. Overall, this article is intended to provide an up-to-date, pedagogical counterpart to the software, which is freely available at https://github.com/Lucy-Forrest-Lab/HDXer.

Abstract 1219: Deep mutational scanning of PDE3A identifies residues required for DNMDP response

Abstract PDE3A-SLFN12 complex formation is induced by a class of compounds, now called “velcrins”, exemplified by the small molecule, DNMDP. Cancer cells that express elevated levels of PDE3A and SLFN12 are sensitive to a velcrin-mediated cytotoxic response, which is independent of PDE3A inhibition. However, the details of complex formation have not yet been revealed. We solved the crystal structure of PDE3A with a series of ligands bound to the active site and found that PDE3A exists as a dimer, and velcrin binding does not cause any obvious structural changes in the PDE3A protein structure. Hydrogen-deuterium exchange (HDX-MS) experiments with velcrin-bound PDE3A in the absence and presence of SLFN12 identified three regions of PDE3A that are shielded from solvent as a result of velcrin-induced SLFN12 binding. Two of these regions are near the velcrin binding site, and the third region lies at the PDE3A homodimerization interface. In order to further investigate the structural relationship between PDE3A, DNMDP, and SLFN12, we took a deep-mutation scanning (DMS) approach to identify residues of PDE3A that impact DNMDP sensitivity. A library of PDE3A alleles was developed in which the sequence encoding amino acids 668-1141, including the PDE3A catalytic domain, was substituted with a codon for every other possible amino acid or a stop codon in the context of the full-length cDNA. The library was transduced into PDE3A-knockout GB1 glioblastoma cells and assessed for survival in the presence of DMSO or DNMDP. Corroborating the HDX-MS data, we identified three regions of PDE3A in which missense mutations abrogated DNMDP response: the active site, the homodimerization surface, and an alpha helix containing amino acid F914. We confirmed that mutations of F914 and the homodimer interface retain the ability to bind resin-conjugated compound but fail to complex with SLFN12. Taken together, the HDX and DMS results suggest that PDE3A dimerization is required to stabilize velcrin-induced SLFN12 binding and implicate the alpha helix containing F914 as the SLFN12 binding interface of PDE3A. Citation Format: Xiaoyun Wu, Malvina Papanastasiou, Gavin Schnitzler, Colin Garvie, Stephanie Hoyt, Terry Zhang, James Mullahoo, Andrew Baker, Joseph McGaunn, Bethany Kaplan, Sooncheol Lee, Martin Lange, Steven Carr, Xiaoping Yang, Federica Piccioni, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. Deep mutational scanning of PDE3A identifies residues required for DNMDP response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1219.

Investigation of the K4[Fe(CN)6]-Mediated Mono- and Bis-Palladium-Catalyzed Cyanation of the Benzothioxanthene Core.

The pallado-catalyzed cyanation of benzothioxanthene imide (BTXI) derivatives is explored herein. Once optimized on the monobromo BTXI, mild reaction conditions were successfully applied to the dibromo derivative affording two regioisomers that have been isolated and structurally solved. Additional hydrogen-deuterium exchange experiments were carried out to support a proposed mechanism involving the formation of a five-membered palladacycle intermediate in the bay area. As well as impacting the structural, photo physical and electrochemical properties of the BTXI core, nitrile moieties were successfully used as orthogonal protecting groups, thus opening doors to new design principles.

Structural and mechanistic bases for a potent HIV-1 capsid inhibitor.

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.