The Role of the Central Globular Domain of Histone H5 in Chromatin Structure

Abstract

Histone H5 contains three tryosines in the central, a polar region of the molecule. All three tryosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain is essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result in a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higherorder chromatin structure.     

The role of the central globular domain of histone H5 in chromatin structure

Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.     

Structural characterization of the trypsin-resistant core in the nuclear sperm-specific protein from Spisula solidissima

Trypsin digestion of the protamine-like protein from Spisula solidissima has revealed the existence of an internal resistant core. The peptide contains 75 amino acid residues, and its primary structure shows some conserved sequences that are common to those found in the core of the somatic histone H5 from chicken erythrocytes. The secondary structure of this core exhibits 33% antiparallel beta-sheet, 18% beta-turns, 37% random coil, and only 10% alpha-helix, in contrast to histone H5. Hydrodynamic measurements indicate a compact globular assembly for the tertiary structure of this peptide, when compared to the more extended shape observed for the whole protein. The possible relatedness of this protein to the histone H1 family is discussed.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The isolation of the globular and non-globular regions of the histone H1 molecule.

Digestion of calf thymus H1 histone with thrombin cleaves the molecule at the sequence -(Pro)-Lys-Lys-Ala-, corresponding to a point approximately 122 residues from the N-terminus (about 56% along the molecule). The N-terminal fragment is shown by proton nuclear magnetic resonance (NMR) to possess the globular structure of the intact histome H1 molecule, whereas the C-terminal fragment appears to possess little or no structure. The N-terminal fragment separates into two peaks on an ion-exchange column, one of which is shown to originate from a single subfraction of calf thymus histone H1 and the other to originate from the other subfractions, by detailed comparison of the NMR spectra. It thus seems that the structure of the H1 histone in solution under physiological conditions consists of a globular head with a highly basic random coil tail. It is suggested that the globular head has a specific binding site on the subunit structure of the chromosome.     

Histone H1 and chromatin higher order structure. Does histone H1 exhibit specific self-association?

1. Histones H1 and H5 in chromatin and in free solution can be cross-linked to higher multimers. Is this due to a specific protein/protein interaction? If so, this interaction might be the structural basis of the condensation of the chromosomal nucleofilament, known to be mediated by histones H1 and H5. 2. Since only the central domain of H1 and H5 exhibits tertiary folding and globular structure, this is the most likely site of specific interaction. 3. Formaldehyde has been used to test whether the central domains of histone H1 from calf thymus or from sea urchin sperm or histone H5 from chicken erythrocytes self-interact. 4. The cross-linking shown by each globular peptide was compared with that of its parent histone. 5. In all three cases the peptide cross-linked to a much lower extent than its intact parent histone and the observed cross-linked rates were roughly in proportion to the relative number of lysine residues parent histone and peptide. 6. It is concluded that there is no specific self-interaction between the globular domains of either H1 or H5 molecules in free solution. 7. This result suggests that specific H1/H1 protein/protein interactions are not the basic cause of chromatin condensation.     

Cooperative binding of the globular domains of histones H1 and H5 to DNA.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.     

The conformation of histone H5 bound to DNA. Maintenance of the globular structure after binding.

Trypsin digestion is used to investigate the conformation of histone H5 when bound to DNA. A central region of H5 comprising residues (22--100) is found to be resistant to digestion and it is concluded that this region is compacted whilst the remaining N- and C-terminal regions are more extended. Since this is the same result found previously for the free solution conformation of histone H5 it follows that a 3-domain structure is preserved on DNA binding. The binding of H5 and the central region (22--100) to DNA is also studied using proton magnetic resonance (270 MHz) and a precipitation approach. It is concluded that all 3 domains of H5 bind to DNA at low ionic strengths. The central domain (residues 22--100) is released at 0.3--0.4 M NaCl, but 0.7 M NaCl is required to release the N- and C-terminal regions. Comparison is made of H5 binding to DNA with that of the related histone H1.     

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome.

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.     

Studies on the interaction of H1 histone with superhelical DNA: characterization of the recognition and binding regions of H1 histones.

The very lysine rich histone, H1, isolated from a variety of sources interacts preferentially with superhelical DNA compared to relaxed DNA duplexes. The nature of this specific interaction has been investigated by studying the ability of various purified fragments of H1 histone from calf thymus to recognize and bind superhelical DNA. The data suggest that the globular region of the H1 histone molecule (amino acid residues 72-106) is involved in the recognition of superhelical DNA. Thus, the H1 histone carboxy-terminal fragment, 72-212, resembles native H1 histone both quantitatively and qualitatively in its ability to discriminate between and bind to superhelical and relaxed DNA while the H1 histone carboxy-terminal fragment, residues 106-212, has lost this specificity, binding superhelical and relaxed DNA equally well. Furthermore, under conditions in which the globular region of the intact H1 histone has been unfolded, the molecule loses its ability to discriminate between superhelical and relaxed DNA, and binds both forms of DNA equally.     

Small-angle X-ray scattering based structure,modeling and molecular dynamics analyses of a family 5 glycoside hydrolase first endo-mannanase named as RfGH5_7 from Ruminococcus flavefaciens

Abstract

Endo-β-1,4-mannanase named as RfGH5_7 from Ruminococcus flavefaciens cloned, expressed and purified earlier was structurally characterized in present study. The RaptorX modeled structure of RfGH5_7 showed a (β/α)₈ Triose-phosphate Isomerase (TIM) barrel fold. The Ramachandran plot assessment of RfGH5_7 showed that all amino acids fall in allowed region except one, Asn22 in the disallowed region. The superposition of RfGH5_7 modeled structure with its nearest homologues revealed that Glu154 acts as proton donor while Glu249 acts as nucleophile. Secondary structure of RfGH5_7 through Circular Dichroism (CD) analysis revealed 33.5% α-helices, 17% β-strands and 49.5% random coils. Molecular Dynamic (MD) simulation showed Root Mean Square Deviation (RMSD), 0.67?nm and radius of gyration (Rg) between 1.9?nm and 1.85?nm. The binding interaction of mannotetraose on the surface of RfGH5_7 structure displayed polar interactions with His219, Tyr221, Trp278, Ser279 and Gly282 residues. Small-angle X-ray scattering (SAXS) analysis displayed the intact and monodispersed nature of the enzyme RfGH5_7. The radius of gyration (Rg) by Guinier analysis for globular shape was found to be 2.29?±?0.09?nm and for rod-shape it was 0.95?±?0.02?nm. Kratky plot confirmed that RfGH5_7 structure is compact and folded in solution. The ab initio derived dummy model of RfGH5_7 displayed single domain structure of yellow humped fish like shape. The RfGH5_7 modeled structure was well fitted with ab initio derived model from SAXS data.

Communicated by Ramaswamy H. Sarma     

Distinct properties of the two putative "globular domains" of the yeast linker histone, Hho1p

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.     

Characteristics of limited trypsinolysis of histone H5 in a solution and as a component of chromatin with different degrees of compactness

Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.     

Co-operative binding of the globular domain of histone H5 to DNA.

The globular domain of histone H5 (GH5) was prepared by trypsin digestion of H5 that was extracted from chicken erythrocyte nuclei with NaCl. Electron microscopy, sucrose gradient centrifugation, native agarose gel electrophoresis and equilibrium density gradient ultracentrifugation show that GH5 binds co-operatively to double-stranded DNA. The electron microscopic images suggest that the GH5-DNA complexes are very similar in structure to co-operative complexes of intact histone H1 (or its variants) with double-stranded DNA, studied previously, which have been proposed to consist of two parallel DNA double helices sandwiching a polymer of the protein. For complexes with GH5 or with intact H1, naked DNA co-sediments with the protein-DNA complexes through sucrose gradients, and DNA also appears to protrude from the ends and sides of the complexes; measurements of the protein-DNA stoichiometry in fractionated samples may not reflect the stoichiometry in the complexes. An estimate of the stoichiometry obtained from the buoyant density of fixed GH5-DNA complexes in CsCl suggests that sufficient GH5 is present in the complexes for the GH5s to be in direct contact, as required by a simple molecular mechanism for the co-operative binding. Chemical crosslinking demonstrates that GH5s are in close proximity in the complexes. In the absence of DNA, GH5-GH5 interactions are weak or non-existent.     

Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The properties of the N-terminal and C-terminal halves of histone H1.

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1--106 and 107--C-terminal. These were studied by proton magnetic resonance and circular dichroism. The N-terminal fragment exhibited some salt-induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C-terminal fragments show very little salt-induced structure. The N-terminal fragments binds to DNA very weakly, but the C-terminal fragment binds as strongly as the whole molecule. In the C-terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 - DNA complexes, and it is suggested that there may be a connection between these phenomena.     

Precise elimination of the N-terminal domain of histone H1.

The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.     

Antibodies against the folding domain of histone H5 cross-react with H1(0) but not with H1

Antibodies to the folding domain (residues 22-100) of histone H5 were elicited in rabbits. Analysis of the specificity of these antibodies by enzyme-linked immunoassay and by diazobenzyloxymethyl cellulose transfer techniques revealed that the antibody cross-reacts strongly with intact H5 and histones H1(0)a and H1(0)b purified from ox liver but not with the four core calf thymus, or with high mobility group proteins. We conclude that the globular region of H5 is serologically homologous to that of H1 degrees and suggest that possible functional similarities between the two proteins reside in this region.     

Two homologous domains of similar structure but different stability in the yeast linker histone, Hho1p

The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.     

Primary, secondary, and tertiary structure of the core of a histone H1-like protein from the sperm of Mytilus

We have analyzed the structure of the trypsin-resistant core of the protein PL-II* of the sperm from Mytilus californianus. The peptide has a molecular mass of 8436 Da and its primary sequence is ATGGAKKP STLSMIVAAIQAMKNRKGSSVQAIRKYILANNKG INTSRLGSAMKLAFAKGLKSGVLVRPKTSAGA SGATGSFRVG. This sequence bears an enormous homology and fulfills the constraints of the consensus sequence of the trypsin-resistant peptides of the proteins of the histone H1 family. Secondary structure analysis using Fourier-transform infared spectroscopy as well as predictive methods indicate the presence of 20-30% beta-structure and approximately 25% alpha-helix for this peptide. As in the case of histone H1 proteins, the protein PL-II* core exhibits a compact globular structure as deduced from hydrodynamic measurements. The presence of a histone H1 protein with protamine-like features, seems to be thus, a common general feature of the chromatin composition in the sperm of the bivalve molluscs.     

Histones H1(0) and H5 share common epitopes with RNA polymerase II

We report here the cross-reaction of RNA polymerase II antiserum with histones H1(0) and H5 and the complementary cross-reactions of antisera to the globular domain of histone H1(0) (GH1(0)) and histone H5 (GH5) with RNA polymerase II. Immunoblotting of RNA polymerase II antiserum with fragments of histone H1(0) localized the cross-reaction at the junction of the globular and C-terminal domains of histone H1(0). The structural homology implied by these cross-reactions is interesting in light of reports that suggest H1(0) may play a role in differentiation and development.     

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber.