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 cyanobacterial cytochrome b6f subunit PetP adopts an SH3 fold in solution.

PetP is a peripheral subunit of the cytochrome b(6)f complex (b(6)f) present in both, cyanobacteria and red algae. It is bound to the cytoplasmic surface of this membrane protein complex where it greatly affects the efficiency of the linear photosynthetic electron flow although it is not directly involved in the electron transfer reactions. Despite the crystal structures of the b(6)f core complex, structural information for the transient regulatory b(6)f subunits is still missing. Here we present the first structure of PetP at atomic resolution as determined by solution NMR. The protein adopts an SH3 fold, which is a common protein motif in eukaryotes but comparatively rare in prokaryotes. The structure of PetP enabled the identification of the potential interaction site for b(6)f binding by conservation mapping. The interaction surface is mainly formed by two large loop regions and one short 310 helix which also exhibit an increased flexibility as indicated by heteronuclear steady-state {(1)H}-(15)N NOE and random coil index parameters. The properties of this potential b(6)f binding site greatly differ from the canonical peptide binding site which is highly conserved in eukaryotic SH3 domains. Interestingly, three other proteins of the photosynthetic electron transport chain share this SH3 fold with PetP: NdhS of the photosynthetic NADH dehydrogenase-like complex (NDH-1), PsaE of the photosystem 1 and subunit α of the ferredoxin-thioredoxin reductase have, similar to PetP, a great impact on the photosynthetic electron transport. Finally, a model is presented to illustrate how SH3 domains modulate the photosynthetic electron transport processes in cyanobacteria.

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.

The structure and conformational switching of Rap1B.

Rap1B is a small GTPase involved in the regulation of numerous cellular processes including synaptic plasticity, one of the bases of memory. Like other members of the Ras family, the active GTP-bound form of Rap1B can bind to a large number of effector proteins and so transmit signals to downstream components of the signaling pathways. The structure of Rap1B bound only to a nucleotide has yet to be solved, but might help reveal an inactive conformation that can be stabilized by a small molecule drug. Unlike other Ras family proteins such as H-Ras and Rap2A, Rap1B crystallizes in an intermediate state when bound to a non-hydrolyzable GTP analog. Comparison with H-Ras and Rap2A reveals conservative mutations relative to Rap1B, distant from the bound nucleotide, which control how readily the protein may adopt the fully activated form in the presence of GTP. High resolution crystallographic structures of mutant proteins show how these changes may influence the hydrogen bonding patterns of the key switch residues.

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.

Conclusive evidence of the reconstituted hexasome proven by native mass spectrometry.

It has been suggested that the hexasome, in which one of the H2A/H2B dimers is depleted from the canonical nucleosome core particle (NCP), is an essential intermediate during NCP assembly and disassembly, but little structural evidence of this exists. In this study, reconstituted products in a conventional NCP preparation were analyzed by native electrospray ionization mass spectrometry, and it was found that the hexasome, which migrated in a manner almost identical to that of the octasome NCP in native polyacrylamide gel electrophoresis, was produced simultaneously with the octasome NCP. This result might contribute to understanding the assembly and disassembly mechanism of NCPs.

Gas-phase structure of the histone multimers characterized by ion mobility mass spectrometry and molecular dynamics simulation.

The minimum structural unit of chromatin is the nucleosome core particle (NCP), consisting of 146 bp of DNA wrapped around a histone octamer, which itself contains two H2A/H2B dimers and one (H3/H4)2 tetramer. These multimers possess functionally important tail regions that are intrinsically disordered. In order to elucidate the mechanisms behind NCP assembly and disassembly processes, which are highly related to gene expression, structural characterization of the H2A/H2B dimer and (H3/H4)2 tetramer will be of importance. In the present study, human histone multimers with disordered tail regions were characterized by electrospray ionization (ESI) ion mobility-mass spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally obtained arrival times of these histone multimer ions showed rather wide distributions, implying that multiple conformers exist for each histone multimer in the gas phase. To examine their structures, MD simulations of the histone multimers were performed first in solution and then in vacuo at four temperatures, resulting in a variety of histone multimer structures. Theoretical collision cross-section (CCS) values calculated for the simulated structures revealed that structural models with smaller CCS values had more compact tail regions than those with larger CCS values. This implied that variation of the CCS values of the histone multimers were primarily due to the random behaviors of the tail regions in the gas phase. The combination of IM-MS and MD simulation enabled clear and comprehensive characterization of the gas-phase structures of histone multimers containing disordered tails.

Native Mass Spectrometry of Protein and DNA Complexes Prepared in Nonvolatile Buffers.

Inorganic salts and nonvolatile-buffer components affect the structure and stability of proteins, and some protein complexes are unable to maintain their function and structure without them. However, it is well-known that these components cause suppression of analyte ionization during the electrospray ionization process. Thus, to establish appropriate methods for observation of the intact ions of protein and DNA complexes by native mass spectrometry (native MS) in the presence of nonvolatile buffer components, we herein examined the effect of ammonium acetate addition to a model homotetramer protein, alcohol dehydrogenase (ADH), which was prepared in a range of nonvolatile buffers, including Tris-HCl, phosphate, and HEPES buffers. Furthermore, native MS of nucleosome core particle (NCP), a large protein-DNA complex, prepared in nonvolatile buffer, was also examined. Intact ADH and NCP ions could be observed upon the addition of ammonium acetate, but NCP does not require as high of a concentration of ammonium acetate as ADH. Well-resolved peaks with different charge numbers could be observed for NCP prepared in Tris-HCl by addition of a lower amount of ammonium acetate than for ADH. This suggests that the effects of additives on native MS of biomolecular complexes can vary depending on the intramolecular interactions present. More specifically, NCP is stabilized mainly by electrostatic interactions, whereas the ADH tetramer depends on the presence of hydrophobic interactions between the four subunits. The results presented herein therefore are expected to contribute to structural biology studies of unstable protein-DNA complexes that are formed transiently during the transcription process.

Solution structure of the isolated histone H2A-H2B heterodimer.

During chromatin-regulated processes, the histone H2A-H2B heterodimer functions dynamically in and out of the nucleosome. Although detailed crystal structures of nucleosomes have been established, that of the isolated full-length H2A-H2B heterodimer has remained elusive. Here, we have determined the solution structure of human H2A-H2B by NMR coupled with CS-Rosetta. H2A and H2B each contain a histone fold, comprising four α-helices and two ß-strands (α1-ß1-α2-ß2-α3-αC), together with the long disordered N- and C-terminal H2A tails and the long N-terminal H2B tail. The N-terminal αN helix, C-terminal ß3 strand, and 310 helix of H2A observed in the H2A-H2B nucleosome structure are disordered in isolated H2A-H2B. In addition, the H2A α1 and H2B αC helices are not well fixed in the heterodimer, and the H2A and H2B tails are not completely random coils. Comparison of hydrogen-deuterium exchange, fast hydrogen exchange, and {(1)H}-(15)N hetero-nuclear NOE data with the CS-Rosetta structure indicates that there is some conformation in the H2A 310 helical and H2B Lys11 regions, while the repression domain of H2B (residues 27-34) exhibits an extended string-like structure. This first structure of the isolated H2A-H2B heterodimer provides insight into its dynamic functions in chromatin.

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities.

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.

Investigation of molecular size of transcription factor TFIIE in solution.

Human general transcription factor IIE (TFIIE), a component of a transcription preinitiation complex associated with RNA polymerase II, was characterized by size-exclusion chromatography, mass spectrometry, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS). Recombinant human TFIIE was purified to homogeneity and shown to contain equimolar amounts of TFIIEalpha (50 kDa) and TFIIEbeta (35 kDa) by SDS-PAGE. In the analysis of size-exclusion chromatography of the purified sample, as already reported, TFIIE was shown to be a 170-kDa alpha(2)beta(2) heterotetramer. However, by using electrospray ionization mass spectrometry the purified sample gave the molecular mass of 84,152 +/- 5, indicating that TFIIE is an alphabeta heterodimer but not a heterotetramer. Analytical ultracentrifugation experiment of TFIIE provided that only a single component with the molecular mass of ca. 80,000 existed in solution, also suggesting an alphabeta heterodimer. In addition, its extraordinarily rod-like molecular shape was confirmed by SAXS. It is likely that the rod-like molecular shape of TFIIE has misled larger molecular size in size-exclusion chromatography, which was calibrated by globular proteins. It is demonstrated that TFIIE exists as a heterodimer under our present conditions in solution, although two molecules of heterodimer might be required for the formation of the preinitiation complex with RNA polymerase II for starting the transcription process.

Charge-neutralization effect of the tail regions on the histone H2A/H2B dimer structure.

It is well known that various modifications of histone tails play important roles in the regulation of transcription initiation. In this study, some lysine (Lys) and arginine (Arg) residues were acetylated and deiminated, respectively, in the histone H2A/H2B dimer, and charge-neutralization effects on the dimer structure were studied by native mass spectrometry. Given that both acetylation and deimination neutralize the positive charges of basic amino acid residues, it had been expected that these modifications would correspondingly affect the gas-phase behavior of the histone H2A/H2B dimer. Contrary to this expectation, it was found that Arg deimination led to greater difficulty of dissociation of the dimer by gas-phase collision, whereas acetylation of Lys residues did not cause such a drastic change in the dimer stability. In contrast, ion mobility-mass spectrometry (IM-MS) experiments showed that arrival times in the mobility cell both of acetylated and of deiminated dimer ions changed little from those of the unmodified dimer ions, indicating that the sizes of the dimer ions did not change by modification. Charge neutralization of Arg, basicity of which is higher than Lys, might have triggered some alteration of the dimer structure that cannot be found in IM-MS but can be detected by collision in the gas phase.

Deimination stabilizes histone H2A/H2B dimers as revealed by electrospray ionization mass spectrometry.

Post-translational modifications of histones for reversibly changing chromosomal structures in promoter regions of genes are a prerequisite for transcriptional activation and repression of genes. Peptidylarginine deiminase 4 (PAD4), which mediates histone deimination by converting arginine residues into citrulline residues, is involved in the repression of gene transcription. However, the mechanism is still unclear. We studied the effects of deimination on the reconstituted histone H2A/H2B dimer structure by electrospray ionization mass spectrometry. Deimination of the H2A/H2B dimer by PAD4 indicated that the mass of H2A increased 2.7 Da, suggesting that two or three Arg residues of H2A were deiminated. Deimination of H2A monomer alone showed a 6.6-Da increase in mass. This indicates that about four more Arg residues of H2A are modified in the monomer state than in the H2A/H2B dimer state. Taking account of the finding that the unstructured portions in proteins are susceptible to deimination by PAD4, it is likely that H2A in the monomer state has a more flexible structure than that in the dimer state. Furthermore, analysis of the association of the H2A/H2B dimer in 2 or 4 M ammonium acetate with nano-electrospray ionization mass spectrometry showed that a modified H2A/H2B dimer was less dissociated into H2A and H2B monomers than an unmodified dimer when high voltages were applied to the sample cone. This study provides convincing evidence that PAD4 deimination stabilizes the histone H2A/H2B dimer.

A protein recycling system for nuclear magnetic resonance-based screening of drug candidates.

A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended.

A novel zinc finger structure in the large subunit of human general transcription factor TFIIE.

The zinc finger domain in the large subunit of TFIIE (TFIIEalpha) is phylogenetically conserved and is essential for transcription. Here, we determined the solution structure of this domain by using NMR. It consisted of one alpha-helix and five beta-strands, showing novel features distinct from previously determined zinc-binding structures. We created point mutants of TFIIEalpha in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Four Zn(2+)-ligand mutants, in which each of cysteine residues at positions 129, 132, 154, and 157 was replaced by alanine, possessed no transcription activities on a linearized template, whereas, on a supercoiled template, interesting functional asymmetry was observed: although the C-terminal two mutants abolished transcription activity (<5%), the N-terminal two mutants retained about 20% activities. The N-terminal two mutants bound stronger to the small subunit of TFIIF than the wild type and the C-terminal two mutants were impaired in their binding abilities to the XPB subunits of TFIIH. These suggest that the structural integrity of the zinc finger domain is essential for the TFIIE function, particularly in the transition from the transcription initiation to elongation and the conformational tuning of this domain for appropriate positioning of TFIIF, TFIIH, and polymerase II would be needed depending on the situation and timing.