Histone H3 lysine 27 crotonylation mediates gene transcriptional repression in chromatin.

Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.

Cryo-electron microscopy structure of the H3-H4 octasome: A nucleosome-like particle without histones H2A and H2B.

The canonical nucleosome, which represents the major packaging unit of eukaryotic chromatin, has an octameric core composed of two histone H2A-H2B and H3-H4 dimers with ∼147 base pairs (bp) of DNA wrapped around it. Non-nucleosomal particles with alternative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Using cryo-electron microscopy, we solved the structure of the H3-H4 octasome, a nucleosome-like particle with a di-tetrameric core consisting exclusively of the H3 and H4 histones. The core is wrapped by ∼120 bp of DNA in 1.5 negative superhelical turns, forming two stacked disks that are connected by a H4-H4' four-helix bundle. Three conformations corresponding to alternative interdisk angles were observed, indicating the flexibility of the H3-H4 octasome structure. In vivo crosslinking experiments detected histone-histone interactions consistent with the H3-H4 octasome model, suggesting that H3-H4 octasomes or related structural features exist in cells.

Solvent effect on the detection of peptides and proteins by nanoelectrospray ionization mass spectrometry: Anomalous behavior of aqueous 2-propanol.

To investigate the solvent effect on the detection of peptides and proteins, nanoelectrospray mass spectra were measured for mixtures of 1 % acetic acid and 5 × 10-6 M gramicidin S (G), ubiquitin (U), and cytochrome c (C) in water (W), methanol (MeOH), 1-propanol (1-PrOH), acetonitrile (AcN), and 2-propanol (2-PrOH). Although doubly protonated G (G2+) and multiply protonated U (Un+) and C (Cn+) were readily detected with a wide range of mixing ratios of W solutions for MeOH, 1-PrOH, and AcN, Cn+ was totally suppressed for the solutions with mixing ratios (v/v) of W/2-PrOH (50/50) and (70/30). However, denatured Cn+ started to be detected with W/2-PrOH (90/10) together with Gn+ (n = 1, 2) and native Un+ (n = 6-8). At the mixing ratio of W/2-PrOH (95/5), native Cn+ (n = 7-10) together with Gn+ (n = 1, 2) and native Un+ (n = 6-8) were detected with high ion intensities. The use of W/2-PrOH (95/5) is profitable because it enables the detection of native proteins with high detection sensitivities.

Single-Cell Native Mass Spectrometry of Human Erythrocytes.

Native mass spectrometry (MS) enables the determination of the molecular mass of protein complexes. Generally, samples for native MS are isolated, purified, and prepared in volatile solutions. However, to understand the function of proteins in living cells, it is essential to characterize the protein complex as is, without isolation/purification of the protein, using the smallest possible amount of the sample. In the present study, we modified the "live single-cell MS" method, which has mainly been used in metabolomics, and applied it to observe hemoglobin directly sampled from human erythrocytes. By optimizing the experimental methods and conditions, we obtained native mass spectra of hemoglobin using only a single erythrocyte, which was directly sampled into a nanoelectrospray ionization emitter using a micromanipulator and microinjector system. That is, our method enables the analysis of ∼0.45 fmol of hemoglobin directly sampled from an erythrocyte. To our knowledge, this is the first report of native MS for endogenous proteins using a single intact human cell.

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study. First, overexpressed DHFR with a His-tag was roughly purified with a Ni-sepharose resin and subjected to native mass spectrometry with or without incubation with an inhibitor, Methotrexate (MTX). Even only with the minimum cleanup by the Ni-sepharose resin, intact ions of DHFR-nicotinamide adenine dinucleotide phosphate (NADPH) and DHFR-NADPH-ligand complexes were successfully observed. By optimizing the preparation procedures of the crude sample for native MS, e.g., avoiding sonication for cell lysis, we successfully observed intact ions of the specific DHFR-NADPH-MTX ternary complex starting with cultivation of E. coli in ≤ 25 mL medium. When the crude DHFR sample was mixed with two, four, or eight candidate compounds, only ions of the specific protein-ligand complex were observed. This indicates that the present system can be used as a rapid and convenient method for the rough determination of binding of specific ligands to the target protein without the time-consuming purification of protein samples. Moreover, it is important to rapidly determine specific interactions with target proteins under conditions similar to those in "real" biological systems. Graphical abstract.

Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system.

Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H-13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.

Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry.

Histone tails, which protrude from nucleosome core particles (NCPs), play crucial roles in the regulation of DNA transcription, replication, and repair. In this study, structural diversity of nucleosomes was investigated in detail by analyzing the observed charge states of nucleosomes reconstituted with various lengths of DNA using positive-mode electrospray ionization mass spectrometry (ESI-MS) and molecular dynamics (MD) simulation. Here, we show that canonical NCPs, having 147 bp DNA closely wrapped around a histone octamer, can be classified into three groups by charge state, with the least-charged group being more populated than the highly charged and intermediate groups. Ions with low charge showed small collision cross sections (CCSs), suggesting that the histone tails are generally compact in the gas phase, whereas the minor populations with higher charges appeared to have more loosened structure. Overlapping dinucleosomes, which contain 14 histone proteins closely packed with 250 bp DNA, showed similar characteristics. In contrast, mononucleosomes reconstituted with a histone octamer and longer DNA (≥250 bp), which have DNA regions uninvolved in the core-structure formation, showed only low-charge ions. This was also true for dinucleosomes with free DNA regions. These results suggest that free DNA regions affect the nucleosome structures. To investigate the possible structures of NCP observed in ESI-MS, computational structural calculations in solution and in vacuo were performed. They suggested that conformers with large CCS values have slightly loosened structure with extended tail regions, which might relate to the biological function of histone tails.

The crystal structure and oligomeric form of Escherichia colil,d-carboxypeptidase A.

Bacterial peptidoglycan is constructed by cross-linking sugar chains carrying pentapeptide building blocks with two d-alanine residues at the C-terminus. Incorporation into the polymer and subsequent breakdown of peptidoglycan releases a tetrapeptide with a single d-alanine residue. Removal of this residue is necessary for the tripeptide to receive a new D-Ala-D-Ala dipeptide in the synthetic pathway, but proteases are generally unable to work with substrates having residues of unusual chirality close to the scissile bond. Processing of the tetrapeptide is carried out by a dedicated ld-carboxypeptidase, which is of interest as a novel drug target. We describe the high resolution crystal structure of the enzyme from E. coli, and demonstrate the dimeric structure is highly conserved.

3D structural analysis of protein O-mannosyl kinase, POMK, a causative gene product of dystroglycanopathy.

Orchestration of the multiple enzymes engaged in O-mannose glycan synthesis provides a matriglycan on α-dystroglycan (α-DG) which attracts extracellular matrix (ECM) proteins such as laminin. Aberrant O-mannosylation of α-DG leads to severe congenital muscular dystrophies due to detachment of ECM proteins from the basal membrane. Phosphorylation at C6-position of O-mannose catalyzed by protein O-mannosyl kinase (POMK) is a crucial step in the biosynthetic pathway of O-mannose glycan. Several mis-sense mutations of the POMK catalytic domain are known to cause a severe congenital muscular dystrophy, Walker-Warburg syndrome. Due to the low sequence similarity with other typical kinases, structure-activity relationships of this enzyme remain unclear. Here, we report the crystal structures of the POMK catalytic domain in the absence and presence of an ATP analogue and O-mannosylated glycopeptide. The POMK catalytic domain shows a typical protein kinase fold consisting of N- and C-lobes. Mannose residue binds to POMK mainly via the hydroxyl group at C2-position, differentiating from other monosaccharide residues. Intriguingly, the two amino acid residues K92 and D228, interacting with the triphosphate group of ATP, are donated from atypical positions in the primary structure. Mutations in this protein causing muscular dystrophies can now be rationalized.

Photoinduced in†Vivo Magnetic Resonance Imaging (MRI) with Rapid CO Release from an MnCO-Protein Needle Composite.

Construction of an artificial protein needle (PN), which includes the membrane puncturing needle domain of bacteriophage T4 conjugated to Mn carbonyl (MnCO) complexes, is reported. The responsiveness to visible light of the MnCO complex makes it useful as a photoinduced in vivo magnetic resonance imaging contrast reagent (MRI CR), because the PN carrier has the potential to deliver the MnCO complex into mouse tumors with retention of coordination structure within the in vivo environment. Moreover, the composite has higher relaxivity and longer circulation as an MRI CR than the corresponding MnCO complex. These results demonstrate construction of a responsive in vivo MRI CR by using an artificial metalloprotein.

Structural analysis and taste evaluation of γ-glutamyl peptides comprising sulfur-containing amino acids.

The structures, flavor-modifying effects, and CaSR activities of γ-glutamyl peptides comprising sulfur-containing amino acids were investigated. The chemical structures, including the linkage mode of the N-terminal glutamic acid, of γ-L-glutamyl-S-(2-propenyl)-L-cysteine (γ-L-glutamyl-S-allyl-L-cysteine) and its sulfoxide isolated from garlic were established by comparing their NMR spectra with those of authentic peptides prepared using chemical methods. Mass spectrometric analysis also enabled determination of the linkage modes in the glutamyl dipeptides by their characteristic fragmentation. In sensory evaluation, these peptides exhibited flavor-modifying effects (continuity) in umami solutions less pronounced but similar to that of glutathione. Furthermore, the peptides exhibited intrinsic flavor due to the sulfur-containing structure, which may be partially responsible for their flavor-modifying effects. In CaSR assays, γ-L-glutamyl-S-methyl-L-cysteinylglycine was most active, which indicates that the presence of a medium-sized aliphatic substituent at the second amino acid residue in γ-glutamyl peptides enhances CaSR activity.

C-terminal acidic domain of histone chaperone human NAP1 is an efficient binding assistant for histone H2A-H2B, but not H3-H4.

Nucleosome assembly protein 1 (NAP1) binds both the (H3-H4)2 tetramer and two H2A-H2B dimers, mediating their sequential deposition on DNA. NAP1 contains a C-terminal acidic domain (CTAD) and a core domain that promotes dimer formation. Here, we have investigated the roles of the core domain and CTAD of human NAP1 in binding to H2A-H2B and H3-H4 by isothermal calorimetry and native mass spectrometry and compared them with the roles of yeast NAP1. We show that the hNAP1 and yNAP1 dimers bind H2A-H2B by two different modes: a strong endothermic interaction and a weak exothermic interaction. A mutant hNAP1, but not yNAP1, dimer lacking CTAD loses the exothermic interaction and shows greatly reduced H2A-H2B binding activity. The isolated CTAD of hNAP1 binds H2A-H2B only exothermically with relatively stronger binding as compared with the exothermic interaction observed for the full-length hNAP1 dimer. Thus, the two CTADs in the hNAP1 dimer seem to provide binding assistance for the strong endothermic interaction of the core domain with H2A-H2B. By contrast, in the relatively weaker binding of hNAP1 to H3-H4 as compared with yNAP1, CTAD of hNAP1 has no significant role. To our knowledge, this is the first distinct role identified for the hNAP1 CTAD.

Comprehensive Characterization of Relationship Between Higher-Order Structure and FcRn Binding Affinity of Stress-Exposed Monoclonal Antibodies.

PURPOSE: In biopharmaceutical development, information regarding higher-order structure (HOS) is important to verify quality and characterize protein derivatives. In this study, we aimed to characterize the association between HOS and pharmacokinetic property of a stress-exposed monoclonal antibody (mAb). METHODS: Purity, primary structure, thermal stability, and HOS were evaluated for mAbs exposed to heat, photo-irradiation, and chemical oxidation. To investigate conformation of stress-exposed mAbs, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was utilized. RESULTS: No distinct difference in secondary or tertiary structure between stress-exposed and non-stressed samples was found by conventional spectroscopic techniques. In binding activity with the neonatal Fc receptor (FcRn), however, a marked decline was observed for force-oxidized mAb and a slight decline was observed for heat- and photodegraded mAbs. Using differential scanning calorimetry, a change in thermal stability was observed in the CH2 domain for all the stress-exposed samples. Using HDX-MS analyses, individual regions with altered conformation could be identified for heat-degraded and force-oxidized samples. CONCLUSIONS: These findings indicate that comprehensive study is important for detecting conformational changes and helpful for predicting biophysical property, and that the evaluation of HOS using several analytical techniques is indispensable for confirming biopharmaceutical quality.

Effect of charge on the conformation of highly basic peptides including the tail regions of histone proteins by ion mobility mass spectrometry.

The first systematic and comprehensive study of the charging behaviour and effect of charge on the conformation of specifically constructed arginine-rich peptides and its significance to the N- and C-terminal basic tail regions of histone proteins was conducted using ion mobility mass spectrometry (IM-MS). Among the basic amino acids, arginine has the greatest impact on the charging behaviour and structures of gas phase ions by virtue of its high proton affinity. A close linear correlation was found between either the maximum charge, or most abundant charge state, that the peptides support and their average collision cross section (CCS) values measured by ion mobility mass spectrometry. The calculated collision cross sections for the lowest energy solution state models predicted by the PEP-FOLD algorithm using a modified MOBCAL trajectory method were found to best correlate with the values measured by IM-MS. In the case of the histone peptides, more compact folded structures, supporting less than the maximum number of charges, were observed. These results are consistent with those previously reported for histone dimers where neutralization of the charge at the basic residues of the tail regions did not affect their CCS values.

Stability of the ßB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.

Ion mobility mass spectrometry was employed to study the structure of the ßB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of ßB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

The crystal structure of the active domain of Anopheles anti-platelet protein, a powerful anti-coagulant, in complex with an antibody.

Blood clotting is a vitally important process that must be carefully regulated to prevent blood loss on one hand and thrombosis on the other. Severe injury and hemophilia may be treated with pro-coagulants, whereas risk of obstructive clotting or embolism may be reduced with anti-coagulants. Anti-coagulants are an extremely important class of drug, one of the most widely used types of medication, but there remains a pressing need for novel treatments, however, as present drugs such as warfarin have significant drawbacks. Nature provides a number of examples of anti-coagulant proteins produced by blood-sucking animals, which may provide templates for the development of new small molecules with similar physiological effects. We have, therefore, studied an Anopheles anti-platelet protein from a malaria vector mosquito and report its crystal structure in complex with an antibody. Overall the protein is extremely sensitive to proteolysis, but the crystal structure reveals a stable domain built from two helices and a turn, which corresponds to the functional region. The antibody raised against Anopheles anti-platelet protein prevents it from binding collagen. Our work, therefore, opens new avenues to the development of both novel small molecule anti-clotting agents and anti-malarials.

Mass spectrometric approach for characterizing the disordered tail regions of the histone H2A/H2B dimer.

The histone H2A/H2B dimer is a component of nucleosome core particles (NCPs). The structure of the dimer at the atomic level has not yet been revealed. A possible reason for this is that the dimer has three intrinsically disordered tail regions: the N- and C-termini of H2A and the N-terminus of H2B. To investigate the role of the tail regions of the H2A/H2B dimer structure, we characterized behaviors of the H2A/H2B mutant dimers, in which these functionally important disordered regions were depleted, using mass spectrometry (MS). After verifying that the acetylation of Lys residues in the tail regions had little effect on the gas-phase conformations of the wild-type dimer, we prepared two histone H2A/H2B dimer mutants: an H2A/H2B dimer depleted of both N-termini (dN-H2A/dN-H2B) and a dimer with the N- and C-termini of H2A and the N-terminus of H2B depleted (dNC-H2A/dN-H2B). We analyzed these mutants using ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS). With IM-MS, reduced structural diversity was observed for each of the tail-truncated H2A/H2B mutants. In addition, global HDX-MS proved that the dimer mutant dNC-H2A/dN-H2B was susceptible to deuteration, suggesting that its structure in solution was somewhat loosened. A partial relaxation of the mutant's structure was demonstrated also by IM-MS. In this study, we characterized the relationship between the tail lengths and the conformations of the H2A/H2B dimer in solution and gas phases, and demonstrated, using mass spectrometry, that disordered tail regions play an important role in stabilizing the conformation of the core region of the dimer in both phases.

Function of homo- and hetero-oligomers of human nucleoplasmin/nucleophosmin family proteins NPM1, NPM2 and NPM3 during sperm chromatin remodeling.

Sperm chromatin remodeling after oocyte entry is the essential step that initiates embryogenesis. This reaction involves the removal of sperm-specific basic proteins and chromatin assembly with histones. In mammals, three nucleoplasmin/nucleophosmin (NPM) family proteins-NPM1, NPM2 and NPM3-expressed in oocytes are presumed to cooperatively regulate sperm chromatin remodeling. We characterized the sperm chromatin decondensation and nucleosome assembly activities of three human NPM proteins. NPM1 and NPM2 mediated nucleosome assembly independently of other NPM proteins, whereas the function of NPM3 was largely dependent on formation of a complex with NPM1. Maximal sperm chromatin remodeling activity of NPM2 required the inhibition of its non-specific nucleic acid-binding activity by phosphorylation. Furthermore, the oligomer formation with NPM1 elicited NPM3 nucleosome assembly and sperm chromatin decondensation activity. NPM3 also suppressed the RNA-binding activity of NPM1, which enhanced the nucleoplasm-nucleolus shuttling of NPM1 in somatic cell nuclei. Our results proposed a novel mechanism whereby three NPM proteins cooperatively regulate chromatin disassembly and assembly in the early embryo and in somatic cells.

Biophysical insights into the dimer formation of human Sirtuin 2.

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild-type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52-357) forms a homodimer in a concentration-dependent manner with a dimer-monomer dissociation constant of 8.3 ± 0.5 µM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non-dimerizing mutants exhibited deacetylation activity for the N-terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292-306, which lacks a SIRT2-specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X-ray structure of the mutant Δ292-306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.

Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.