Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.     

Histones and nucleosomes in Archaea and Eukarya: a comparative analysis

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3+H4)₂ tetramer. Received: January 22, 1998 / Accepted: February 16, 1998     

Both DNA and histone fold sequences contribute to archaeal nucleosome stability.

The roles and interdependence of DNA sequence and archaeal histone fold structure in determining archaeal nucleosome stability and positioning have been determined and quantitated. The presence of four tandem copies of TTTAAAGCCG in the polylinker region of pLITMUS28 resulted in a DNA molecule with increased affinity (DeltaDeltaG of approximately 700 cal mol(-1)) for the archaeal histone HMfB relative to the polylinker sequence, and the dominant, quantitative contribution of the helical repeats of the dinucleotide TA to this increased affinity has been established. The rotational and translational positioning of archaeal nucleosomes assembled on the (TTTAAAGCCG)(4) sequence and on DNA molecules selectively incorporated into archaeal nucleosomes by HMfB have been determined. Alternating A/T- and G/C-rich regions were located where the minor and major grooves, respectively, sequentially faced the archaeal nucleosome core, and identical positioning results were obtained using HMfA, a closely related archaeal histone also from Methanothermus fervidus. However, HMfA did not have similarly high affinities for the HMfB-selected DNA molecules, and domain-swap experiments have shown that this difference in affinity is determined by residue differences in the C-terminal region of alpha-helix 3 of the histone fold, a region that is not expected to directly interact with DNA. Rather this region is thought to participate in forming the histone dimer:dimer interface at the center of an archaeal nucleosome histone tetramer core. If differences in this interface do result in archaeal histone cores with different sequence preferences, then the assembly of alternative archaeal nucleosome tetramer cores could provide an unanticipated and novel structural mechanism to regulate gene expression.     

Conserved eukaryotic histone-fold residues substituted into an archaeal histone increase DNA affinity but reduce complex flexibility

Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.     

Core histones of the amitochondriate protist, Giardia lamblia

Genes coding for the core histones H2a, H2b, H3, and H4 of Giardia lamblia were sequenced. A conserved organism- and gene-specific element, GRGCGCAGATTTVGG, was found upstream of the coding region in all core histone genes. The derived amino acid sequences of all four histones were similar to their homologs in other eukaryotes, although they were among the most divergent members of this protein family. Comparative protein structure modeling combined with energy evaluation of the resulting models indicated that the G. lamblia core histones individually and together can assume the same three-dimensional structures that were established by X-ray crystallography for Xenopus laevis histones and the nucleosome core particle. Since G. lamblia represents one of the earliest-diverging eukaryotes in many different molecular trees, the structure of its histones is potentially of relevance to understanding histone evolution. The G. lamblia proteins do not represent an intermediate stage between archaeal and eukaryotic histones.     

Mutational analysis of differences in thermostability between histones from mesophilic and hyperthermophilic archaea

Amino acid residues responsible for the large difference in thermostability between HMfB and HFoB, archaeal histones from the hyperthermophile Methanothermus fervidus and the mesophile Methanobacterium formicicum, respectively, have been identified by site-specific mutagenesis. The thermal denaturation of approximately 70 archaeal histone variants has been monitored by circular dichroism, and the data generated were fit to a two-state unfolding model (dimer-->two random coil monomers) to obtain a standard-state (1M) melting temperature for each variant dimer. The results of single-, double-, and triple-residue substitutions reveal that the much higher stability of rHMfB dimers, relative to rHFoB dimers, is conferred predominantly by improved intermolecular hydrophobic interactions near the center of the histone dimer core and by additional favorable ion pairs on the dimer surface.     

Archaeal histone selection of nucleosome positioning sequences and the procaryotic origin of histone-dependent genome evolution

Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.     

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.     

Unique fluorophores in the dimeric archaeal histones hMfB and hPyA1 reveal the impact of nonnative structure in a monomeric kinetic intermediate

Homodimeric archaeal histones and heterodimeric eukaryotic histones share a conserved structure but fold through different kinetic mechanisms, with a correlation between faster folding/association rates and the population of kinetic intermediates. Wild-type hMfB (from Methanothermus fervidus) has no intrinsic fluorophores; Met35, which is Tyr in hyperthermophilic archaeal histones such as hPyA1 (from Pyrococcus strain GB-3A), was mutated to Tyr and Trp. Two Tyr-to-Trp mutants of hPyA1 were also characterized. All fluorophores were introduced into the long, central alpha-helix of the histone fold. Far-UV circular dichroism (CD) indicated that the fluorophores did not significantly alter the helical content of the histones. The equilibrium unfolding transitions of the histone variants were two-state, reversible processes, with DeltaG degrees (H2O) values within 1 kcal/mol of the wild-type dimers. The hPyA1 Trp variants fold by two-state kinetic mechanisms like wild-type hPyA1, but with increased folding and unfolding rates, suggesting that the mutated residues (Tyr-32 and Tyr-36) contribute to transition state structure. Like wild-type hMfB, M35Y and M35W hMfB fold by a three-state mechanism, with a stopped-flow CD burst-phase monomeric intermediate. The M35 mutants populate monomeric intermediates with increased secondary structure and stability but exhibit decreased folding rates; this suggests that nonnative interactions occur from burial of the hydrophobic Tyr and Trp residues in this kinetic intermediate. These results implicate the long central helix as a key component of the structure in the kinetic monomeric intermediates of hMfB as well as the dimerization transition state in the folding of hPyA1.     

A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et al., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using gel shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nucleotides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 A resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five alpha-helices and a four stranded beta-sheet. The beta-sheet is sandwiched by three helices (alpha1, alpha4, and alpha5) at one side and two helices (alpha2 and alpha3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function.     

Crystal structure of protein Ph1481p in complex with protein Ph1877p of archaeal RNase P from Pyrococcus horikoshii OT3: implication of dimer formation of the holoenzyme

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.     

The crystal structure of Aq_328 from the hyperthermophilic bacteria Aquifex aeolicus shows an ancestral histone fold

The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 A by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 A over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein's thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper.     

Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

An archaeal histone-like protein as an efficient DNA carrier in gene transfer

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNA was 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.     

Molecular components of the archaeal nucleosome

Here we describe the organization of the archaeal nucleosome, in which four archaeal histones are circumscribed by approximately 80 bp of DNA. Through a combination of sequence comparisons, 3D structural studies, site-directed mutagenesis and assays for DNA binding, we have assigned functions to most of the individual residues in the histone fold of the representative archaeal histone rHMfB. By SELEX selection, the sequences of DNA molecules that are most readily bound and wrapped by rHMfB into archaeal nucleosomes in vitro have been identified, and these define DNA structures that position archaeal nucleosome assembly.     

Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

The N-terminal tails of the H2A-H2B histones affect dimer structure and stability

The histone proteins of the core nucleosome are highly basic and form heterodimers in a "handshake motif." The N-terminal tails of the histones extend beyond the canonical histone fold of the hand-shake motif and are the sites of posttranslational modifications, including lysine acetylations and serine phosphorylations, which influence chromatin structure and activity as well as alter the charge state of the tails. However, it is not well understood if these modifications are signals for recruitment of other cellular factors or if the removal of net positive charge from the N-terminal tail plays a role in the overall structure of chromatin. To elucidate the effects of the N-terminal tails on the structure and stability of histones, the highly charged N-terminal tails were truncated from the H2A and H2B histones. Three mutant dimers were studied: DeltaN-H2A/WT H2B; WT H2A/DeltaN-H2B, and DeltaN-H2A/DeltaN-H2B. The CD spectra, stabilities to urea-denaturation, and the salt-dependent stabilization of the three truncated dimers were compared with those of the wild-type dimer. The data support four conclusions regarding the effects of the N-terminal tails of H2A and H2B: (1) Removal of the N-terminal tails of H2A and H2B enhance the helical structure of the mutant heterodimers. (2) Relative to the full-length WT heterodimer, the DeltaN-H2A/WT H2B dimer is destabilized, while the WT H2A/DeltaN-H2B and DeltaN-H2A/DeltaN-H2B dimers are slightly stabilized. (3) The truncated dimers exhibit decreased m values, relative to the WT dimer, supporting the hypothesis that the N-terminal tails in the isolated dimer adopt a collapsed structure. (4) Electrostatic repulsion in the N-terminal tails decreases the stability of the H2A-H2B dimer.