Protein oxidative footprinting, using hydroxyl radical labeling detected by bottom-up proteomics, has progressed from an emerging method to a widely used approach in structural biology. Hydroxyl radicals generated from hydrogen peroxide (via photolysis, Fenton chemistry or electrochemistry) or directly from water (via X-rays, plasma or gamma rays) irreversibly encode structural information within protein side chains, which is read out using standard liquid chromatography-mass spectrometry workflows. Quantitative changes in labeling report on solvent accessibility and reveal effects of protein-protein interactions, ligand binding, protein folding, conformational changes or applied stress. Comparing labeling patterns between states provides detailed maps of structural changes and interaction sites. Over the past decade, oxidative footprinting has proven valuable as a solution-phase and in-cell method for protein structure analysis. This Perspective summarizes best practices for experimental design, sample processing, data analysis, interpretation and integration with orthogonal data, offering a consensus framework to guide application of oxidative footprinting in academic and biopharmaceutical research.

Maintenance of S-adenosylmethionine (SAM) homeostasis is essential for methylation of biomolecules, nucleotide and polyamine synthesis, and redox homeostasis. While all methyltransferases consume SAM, only a subset of highly tissue specific methyltransferases regulates SAM homeostasis. Among them, glycine N-methyltransferase (GNMT) is enriched in the liver and its dysregulated activity has been linked to compromised liver function. GNMT is inhibited by the methyl carrier 5-methyltetrahydrofolate (5mTHF), suggesting a negative-feedback mechanism regulating its activity. Here, we identify the GNMT N-terminal tail, and specifically phosphorylation at serine 9 (S9ph), as a regulatory modification linking folate-dependent feedback inhibition to SAM homeostasis. Structural and biochemical analyses and molecular dynamics simulations revealed that the N-terminal tail is required for catalytic turnover of SAM and for 5mTHF binding. Phosphoproteomic analysis showed that GNMT S9ph is abundant in mouse liver and further enriched in aged mice. Consistent with loss of folate-dependent negative feedback, both distal N-terminal truncation (residues 1-8) and a phosphomimetic substitution abolished 5mTHF binding while maintaining catalytic activity. In hepatocyte cell lines lacking endogenous GNMT, lentiviral overexpression of constitutively active GNMT mutants depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced transcriptional responses consistent with methyl-donor stress. Together, these findings identify the GNMT N-terminus as a tunable phosphoregulatory domain that dynamically regulates GNMT activity and cellular methylation potential.

Maintenance of S-adenosylmethionine (SAM) homeostasis is essential for methylation of biomolecules, nucleotide and polyamine synthesis, and redox balance. While all methyltransferases consume SAM, only a subset of highly tissue specific methyltransferases regulate methylation potential. Among them, glycine N-methyltransferase (GNMT) is enriched in the liver and its dysregulated activity has been linked to compromised liver function. GNMT is inhibited by the methyl carrier 5-methyltetrahydrofolate (5mTHF), suggesting a negative-feedback mechanism regulating its activity. Here, we identify the GNMT N-terminal tail, and specifically phosphorylation at serine 9 (S9ph), as a regulatory modification linking folate-dependent feedback inhibition to SAM homeostasis. Structural and biochemical analyses and molecular dynamics simulations revealed that the N-terminal tail is required for catalytic turnover of SAM and for 5mTHF binding. Phosphoproteomic analysis showed that GNMT S9ph is abundant in mouse liver and further enriched in aged mice. Consistent with loss of folate-dependent negative feedback, both distal N-terminal truncation (residues 1-8) and a phosphomimetic substitution abolished 5mTHF binding while maintaining catalytic activity. In hepatocyte cell lines lacking endogenous GNMT, lentiviral overexpression of constitutively active GNMT mutants depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced transcriptional responses consistent with methyl-donor stress. Together, these findings identify the GNMT N-terminus as a tunable phosphoregulatory domain that dynamically regulates GNMT activity and cellular methylation potential.

Characterizing protein-ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone. These results highlight the value of combining these methods to compare compounds in drug discovery and development. To better understand the structural context of the HDX-MS and OX-MS drug-dependent perturbations, we mapped these data on Bcl-2-Venetoclax and Bcl-2-S55746 cocrystal structures and compared these results with the structure of apo Bcl-2. HDX-MS shows that Venetoclax more strongly impacts the protein backbone compared to S55746. OX-MS reveals oxidation perturbations rationalized by direct side-chain protection as well as by crystallographically observed drug-induced protein restructuring. Both methods report the perturbation of some, but not all, residues mapped within 4 Å of the bound drugs in the crystal structures. Concordant characterization of backbone and side-chain accessibility will enhance our understanding of in-solution protein structure dynamics and protein-ligand interactions during drug discovery, development, and characterization, particularly when high-resolution structures are lacking.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1α and nucleosomes. We have elucidated the cryo-EM structure of an HP1α dimer bound to an H2A.Z nucleosome, revealing that the HP1α dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1α functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.

DNA modifications are small covalent chemical groups that modify nucleotides to regulate DNA readout. Anomalous abundance and genome-wide localization of these modifications can negatively tune gene expression and propagate into unbalanced epigenetics regulation, which is known to be associated with multiple conditions such as cancer, diabetes and aging. We present a direct injection mass spectrometry (DI-MS) platform that offers fast, accurate and precise quantitation of global levels of DNA cytidine methylation (mC) and hydroxymethylation (hmC) in less than one minute per sample. On the contrary to most methods adopting mass spectrometry for the analysis of nucleotide modifications, in this DI-MS approach we eliminate the use of liquid chromatography, increasing throughput, eliminating issues of carryover and batch effects caused by column contamination across samples. In addition, potential biases in detection efficiency of modified nucleotides with different binding efficiency to stationary phases is eliminated, as no chromatographic separation is adopted. This method can analyze >1000 samples per day, overcoming the throughput of next-generation sequencing.•Direct injection mass spectrometry improves throughput and precision compared to liquid chromatography.•Direct injection can be used to quantify in less than one minute global levels of DNA methylation and hydroxymethylation.•The unbiased acquisition can be potentially utilized to analyze other nucleotide modifications.

Coronavirus disease 2019 ( COVID-19 ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( S ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F ™ and ExpiCHO-S ™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%-7% (R2 > 0.98) and 0.13%-1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC50 values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication. HIV-1 integrase (IN) binds the host factor INI1/SMARCB1, which is required at multiple stages of HIV-1 replication. Here, the authors show that the same IN residues are involved in INI1 and RNA binding and in influencing particle morphogenesis and suggest that the IN-binding INI1 domain is structurally similar to HIV TAR RNA.

S100A12 is a member of the Ca(II) binding S100 family of proteins that functions within the human innate immune system. As a metal‐chelator, S100A12 binds both Ca(II) and Zn(II) at structurally distinct sites. Ca(II) binding enhances the Zn(II) binding affinity and the mechanism of the mode of action of Ca(II) is not fully understood. Here, using solution and solid state NMR spectroscopy, we have conducted structural characterization of S100A12 upon Ca(II) and Zn(II) binding. We demonstrate that distinct conformational changes are introduced in helix II and the hinge domain of the protein upon Zn(II) binding to Ca(II)‐S100A12, suggesting that both ions modulate the structure of S100A12. Moreover, Ca(II)‐S100A12 exhibits a broader pH range for Zn(II) binding compared to the apo protein by restricting conformational changes to Zn(II) binding residue, D25. In conclusion, our results indicate that Ca(II) stabilizes Zn(II) binding affinity by modulating structural changes. These findings will be of value for understanding of the nutrient‐sequestration effect of neutrophils and the interaction between the S100A12 and other receptors such as receptor for advanced glycation endproducts (RAGE) and toll‐like receptor 4 (TLR).Support or Funding InformationThis work was supported by the City University of New York startup funds, the National Institutes of Health (R15GM131338) and PSC CUNY award (62234‐00 50) to Rupal Gupta. NMR data presented herein were collected (in part) at the City University of New York’s Advanced Science Research Center (CUNY ASRC) Biomolecular NMR Facility.MAS NMR spectra of apo and Zn2+‐S100A12. a) Left: DARR (14.1 T) and NCACX (16.4 T) 2D spectra apo S100A12 with resonance assignments; Right: strip plots from CBCACONH (blue) and HNCACB (orange) solution NMR spectra for residues T26‐S28. Signals belonging to an assigned residue in MAS and solution spectra are connected with the same color; b) overlay of Zn2+‐(orange) and apo‐(blue) S100A12 DARR spectra acquired at 14.1 T and; c)–e) expansions of b) with assignments of the perturbed residues.Figure 1

S100A12 is a member of the Ca2+ binding S100 family of proteins that functions within the human innate immune system. Zinc sequestration by S100A12 confers antimicrobial activity when the protein is secreted by neutrophils. Here, we demonstrate that Ca2+ binding to S100A12's EF-hand motifs and Zn2+ binding to its dimeric interface cooperate to induce reversible self-assembly of the protein. Solution and magic angle spinning nuclear magnetic resonance spectroscopy on apo-, Ca2+-, Zn2+-, and Ca2+,Zn2+-S100A12 shows that significant metal binding-induced chemical shift perturbations, indicative of conformational changes, occur throughout the polypeptide chain. These perturbations do not originate from changes in the secondary structure of the protein, which remains largely preserved. While the overall structure of S100A12 is dominated by Ca2+ binding, Zn2+ binding to Ca2+-S100A12 introduces additional structural changes to helix II and the hinge domain (residues 38-53). The hinge domain of S100A12 is involved in the molecular interactions that promote chemotaxis for human monocyte, acute inflammatory responses and generates edema. In Ca2+-S100A12, helix II and the hinge domain participate in binding with the C-type immunoglobulin domain of the receptor for advanced glycation products (RAGE). We discuss how the additional conformational changes introduced to these domains upon Zn2+ binding may also impact the interaction of S100A12 and target proteins such as RAGE.

BACKGROUND:For almost four decades, hydroxyl radical chemically generated by Fenton chemistry has been a mainstay for the oxidative 'footprinting' of macromolecules. OBJECTIVE:In this article, we start by reviewing the application of chemical generation of hydroxyl radical to the development of oxidative footprinting of DNA and RNA and the subsequent application of the method to oxidative footprinting of proteins. We next discuss a novel strategy for generating hydroxyl radicals by Fenton chemistry that immobilizes catalytic iron on a solid surface (Pyrite Shrink Wrap laminate) for the application of nucleic acid and protein footprinting. METHOD:Pyrite Shrink-Wrap Laminate is fabricated by depositing pyrite (Fe-S2, aka 'fool's gold') nanocrystals onto thermolabile plastic (Shrinky Dink). The laminate can be thermoformed into a microtiter plate format into which samples are deposited for oxidation. RESULTS:We demonstrate the utility of the Pyrite Shrink-Wrap Laminate for the chemical generation of hydroxyl radicals by mapping the surface of the T-cell co-stimulatory protein Programmed Death - 1 (PD-1) and the interface of the complex with its ligand PD-L1. CONCLUSION:We have developed and validated an affordable and reliable benchtop method of hydroxyl radical generation that will broaden the application of protein oxidative footprinting. Due to the minimal equipment required to implement this method, it should be easily adaptable by many laboratories with access to mass spectrometry.

Fusion of host and viral membranes is a crucial step during infection by enveloped viruses. In the structurally-defined "class I″ viral glycoproteins, the formation of a highly stable α-helical bundle by the ectodomain of the fusion subunit (e.g., GP2 for Marburg virus, MARV) is postulated to provide the energetic driving force to overcome barriers associated with membrane fusion. Upon cell binding, the fusion subunit is proposed to form an extended intermediate that bridges both the viral and host membranes, and collapse of this extended intermediate brings the two membranes into proximity. While there is much high-resolution structural data available for prefusion and post-fusion structures of viral glycoproteins, little information is available about intermediate conformations especially in the context of the fusion loop/peptide (FL or FP) and membrane-proximal external region (MPER)/transmembrane (TM) segments. We present structural and functional studies on segments of MARV GP2 that encompass the FL and MPER/TM in detergent micelles and lipid bicelles. A protein that contains most elements of GP2 ("MGP2-full") is α-helical in membrane-mimicking environments and has pH-dependent membrane lytic activity. MGP2-full is monomeric under such conditions, contrasting with the trimeric species that has been described previously for MARV GP2 ectodomain in aqueous buffer. Variants of MARV GP2 containing the N- and C-terminal halves ("MGP2-FNL" and "MGP2-CMT", respectively) have similar properties. This work provides novel insight into conformational and membrane-perturbing properties of the MARV fusion subunit and how they may relate to viral membrane fusion.

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from Drosophila melanogaster. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel β-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family.

Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2-5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy. miRFPs enable development of several types of NIR biosensors, such as for protein-protein interactions, RNA detection, signalling cascades and cell fate. We demonstrate this by engineering the monomeric fluorescence complementation reporters, the IκBα reporter for NF-κB pathway and the cell cycle biosensor for detection of proliferation status of cells in culture and in animals. miRFPs allow non-invasive visualization and detection of biological processes at different scales, from super-resolution microscopy to in vivo imaging, using the same probes.