DNA-induced Secondary Structure of the Carboxyl-terminal Domain of Histone H1

Abstract

We have studied the secondary structure of the carboxyl-terminal domains of linker histone H1 subtypes H10 (C-H10) and H1t (C-H1t), free in solution and bound to DNA, by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxyl-terminal domain include the α-helix, β-structure, turns, and open loops. The structure of the bound domain shows a significant dependence on salt concentration. In low salt (10 mm NaCl), there is a residual amount of random coil, 7% in C-H10 and 12% in C-H1t. In physiological salt concentrations (140 mm NaCl), the carboxyl termini become fully structured. Under these conditions, C-H10 contained 24% α-helix, 25% β-structure, 17% open loops, and 33% turns. The latter component could include a substantial proportion of the 310 helix. Despite their low sequence identity (∼30%), the representation of the different structural motifs in C-H1t was similar to that in C-H10. Examination of the changes in the amide I components in the 20–80 °C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding. We have studied the secondary structure of the carboxyl-terminal domains of linker histone H1 subtypes H10 (C-H10) and H1t (C-H1t), free in solution and bound to DNA, by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but becomes extensively folded upon interaction with DNA. The secondary structure elements present in the bound carboxyl-terminal domain include the α-helix, β-structure, turns, and open loops. The structure of the bound domain shows a significant dependence on salt concentration. In low salt (10 mm NaCl), there is a residual amount of random coil, 7% in C-H10 and 12% in C-H1t. In physiological salt concentrations (140 mm NaCl), the carboxyl termini become fully structured. Under these conditions, C-H10 contained 24% α-helix, 25% β-structure, 17% open loops, and 33% turns. The latter component could include a substantial proportion of the 310 helix. Despite their low sequence identity (∼30%), the representation of the different structural motifs in C-H1t was similar to that in C-H10. Examination of the changes in the amide I components in the 20–80 °C temperature interval showed that the secondary structure of the DNA-bound C-H1t is for the most part extremely stable. The H1 carboxyl-terminal domain appears to belong to the so-called disordered proteins, undergoing coupled binding and folding. H1 linker histones are thought to be primarily responsible for the condensation of the thick chromatin fiber. It is currently accepted that histone H1 could have a regulatory role in transcription through the modulation of chromatin higher order structure. H1 has been described as a general transcriptional repressor because it contributes to chromatin condensation, which limits the access of the transcriptional machinery to DNA. However, H1 may regulate transcription at a more specific level, participating in complexes that either activate or repress specific genes (1Zlatanova J. Van Holde K. J. Cell Sci. 1992; 103: 889-895Crossref PubMed Google Scholar, 2Khochbin S. Wolffe A.P. Eur. J. Biochem. 1994; 225: 501-510Crossref PubMed Scopus (99) Google Scholar, 3Bouvet L. Dimitrov S. Wolffe A.P. Genes Dev. 1994; 8: 1147-1159Crossref PubMed Scopus (210) Google Scholar, 4Shen X. Gorovsky M.A. Cell. 1996; 86: 475-483Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 5Wolffe A.P. Khochbin P.S. Dimitrov S. 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In mammals, six somatic subtypes (designated H1a–H1e and H10), a male germ line-specific subtype (H1t), and an oocyte-specific subtype (H1oo) have been identified (11Panyim S. Chalkley R. Biochem. Biophys. Res. Commun. 1969; 37: 1042-1049Crossref PubMed Scopus (239) Google Scholar, 12Bucci L.R. Brock W.A. Meistrich M.L. Exp. Cell Res. 1982; 140: 111-118Crossref PubMed Scopus (83) Google Scholar, 13Lennox R.W. J. Biol. Chem. 1984; 259: 669-672Abstract Full Text PDF PubMed Google Scholar, 14Tanaka M. Hennebold J.D. Macfarlane J. Adashi E.Y. Development (Camb.). 2001; 128: 655-664Crossref PubMed Google Scholar). The subtypes differ in timing of expression (15Khochbin S. Gene (Amst.). 2001; 27: 11-12Google Scholar), extent of phosphorylation (16Lennox R.W. Oshima R.G. Cohen L.H. J. Biol. Chem. 1982; 257: 5183-5189Abstract Full Text PDF PubMed Google Scholar), turnover rate (17Hall J.M. Cole R.D. Biochemistry. 1985; 24: 7765-7771Crossref PubMed Scopus (15) Google Scholar, 18Domínguez V. Piña B. Suau P. Development (Camb.). 1992; 115: 181-185PubMed Google Scholar), binding affinity (19Th'ng J.P.H. Sung R. Ye M. Hendzel M.J. J. Biol. Chem. 2005; 280: 27809-27814Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar), and evolutionary stability (20Ponte I. Vidal-Taboada J.M. Suau P. Mol. Biol. Evol. 1998; 15: 702-708Crossref PubMed Scopus (68) Google Scholar). Differences in DNA condensing capacity have also been demonstrated for some subtypes (21Liao L.W. Cole R.D. J. Biol. Chem. 1981; 256: 10124-10128Abstract Full Text PDF PubMed Google Scholar, 22Khadake J.R. Rao M.R. Biochemistry. 1995; 34: 15792-15801Crossref PubMed Scopus (90) Google Scholar, 23Talasz H. Sapojnikova N. Helliger W. Linder H. Puschendorf B. J. Biol. Chem. 1998; 273: 32236-32243Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Linker histones contain three distinct domains: a short amino-terminal domain (20–35 amino acids), a central globular domain (∼80 amino acids, consisting of a helix bundle and a β-hairpin), and a long carboxyl-terminal domain (∼100 amino acids) (24Hartman P.G. Chapman G.E. Moss T. Bradbury E.M. Eur. J. Biochem. 1977; 77: 45-51Crossref PubMed Scopus (229) Google Scholar). The amino- and carboxyl-terminal domains are highly basic. The distribution of charge in the carboxyl-terminal domain is extremely uniform despite the variation in sequence in the different subtypes (25Subirana J.A. Biopolymers. 1990; 29: 1351-1357Crossref PubMed Scopus (44) Google Scholar). The carboxyl-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo (19Th'ng J.P.H. Sung R. Ye M. Hendzel M.J. J. Biol. Chem. 2005; 280: 27809-27814Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 26Hendzel M.J. Lever M.A. Crawford E. Th'ng J.P.H. J. Biol. Chem. 2004; 279: 20028-20034Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Several studies indicate that the ability of linker histones to stabilize chromatin folding resides in the carboxyl-terminal domain of the protein (27Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Allan J. Hartman P.G. Crane-Robinson C. Avilés F.X. Nature. 1980; 288: 675-679Crossref PubMed Scopus (526) Google Scholar, 29Allan J. Mitchel T. Harborne N. Bohm L. Crane-Robinson C. J. Mol. Biol. 1986; 187: 591-601Crossref PubMed Scopus (263) Google Scholar). The preferential binding of histone H1 to scaffold-associated regions appears to be determined by the carboxyl-terminal domain (30Roque A. Orrego M. Ponte I. Suau P. Nucleic Acids Res. 2004; 32: 6111-6119Crossref PubMed Scopus (31) Google Scholar). The carboxyl-terminal domain is also responsible for the activation of apoptotic nuclease (31Widlak P. Kalinowska M. Parseghian M.H. Lu X. Hansen J.C. Garrard W.T. Biochemistry. 2005; 44: 7871-7878Crossref PubMed Scopus (54) Google Scholar). Knowledge of the structure of the carboxyl-terminal domain of H1 once bound to DNA is thus important to our understanding of H1 function. The amino- and carboxyl-terminal domains have little defined structure in solution, although they may contain a considerable amount of turn-like conformations in rapid equilibrium with unfolded states. Molecular modeling (32Bharath M.M. Chandra N.R. Rao M.R. Proteins. 2002; 49: 71-81Crossref PubMed Scopus (33) Google Scholar) and induction of α-helix in the presence of secondary structure stabilizers (33Clark D.J. Hill C.S. Martin S.R. Thomas J.O. EMBO J. 1988; 7: 69-75Crossref PubMed Scopus (136) Google Scholar), such as 2,2,2-trifluoroethanol (TFE) 2The abbreviations used are: TFE, 2,2,2-trifluoroethanol; C-H10, carboxyl-terminal domain of histone H10; C-H1t, carboxyl-terminal domain of histone H1t. and HClO4, suggest that the carboxyl termini would acquire a folded conformation on binding to DNA. We have shown previously that interaction with DNA induces stable helix and turn structures in a 23-residue peptide from the H10 carboxyl terminus (CH-1, residues 99–121, a peptide studied previously (34Vila R. Ponte I. Jiménez M.A. Rico M. Suau P. Protein Sci. 2000; 9: 1-10PubMed Google Scholar, 35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar)). This region, adjacent to the globular domain, is shown to behave as a specific subdomain in the stabilization of folded chromatin structures by histone H1 (27Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Inducible helical elements have also been characterized in the amino-terminal domains of H10 and H1e (36Vila R. Ponte I. Collado M. Arrondo J.L.R. Jiménez M.A. Rico M. Suau P. J. Biol. Chem. 2001; 276: 46429-46435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 37Vila R. Ponte I. Jiménez M.A. Rico M. Suau P. Protein Sci. 2001; 11: 214-220Crossref Scopus (29) Google Scholar). In the present study we have used infrared spectroscopy to study the secondary structure of the carboxyl-terminal domains of H10 and H1t in aqueous solution and in the complexes with DNA. The carboxyl-terminal domains have little structure in solution but become fully structured upon interaction with DNA in physiological salt (140 mm NaCl). The secondary structure elements present in the complexes include α-helix, β-structure, turns, and open loops. The study of the entire carboxyl-terminal domain has provided new information, not readily accessible to studies with peptides, on the variety of structural motifs present in the DNA-bound domain, the salt dependence and thermal stability of the folded structure, and the conservation of the protein structure among divergent subtypes. It has also shown that the property of coupled binding and folding, already demonstrated for the previously studied CH-1 peptide (35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), is shared by the entire domain. Cloning, Expression, and Purification of the Carboxyl-terminal Domains of Histone H1 Subtypes—The sequences encoding the carboxyl-terminal domains of histones H10 and H1t were cloned and expressed. All gene fragments were amplified from mouse genomic DNA by PCR. The primers were 5′-CCACCATGGATGAGCCTAAAAGGT-3′ and 5′-GGAGATCTTTACTTCTTCTTGCTGGCCCTCT-3′ for the carboxyl-terminal domain of H10 and 5′-GTACCATGGCGGCTTCAGGGAACGAC-3′ and 5′-ACGGATCCTTACTTCCTCCCTGCTGCCTTCCT-3′ for the carboxyl-terminal domain of H1t. The amplification products were cloned in the pQE60 vector (Qiagen) using the NcoI and BglII restriction sites to yield the expression vectors pCTH10 and pCTH1t. The recombinant plasmids were transformed into Escherichia coli M15 (Qiagen). Cells were grown to an of and with the expression to for at Cells were in mm for at The was at for The were on a with with The bound to the recombinant proteins, was with mm the were by through for spectroscopy were carboxyl-terminal domain in mm in aqueous solution and in were were on a in mm of at The were with and as The helical was from the at to the of Biochemistry. PubMed Scopus Google helical is the of peptide carboxyl-terminal domains of histone H1 were at in mm or mm complexes contained mouse DNA and the amount of were on a with a using a with and for and for for and were and the were with a of at and have been described previously J.L.R. Biophys. Mol. Biol. 1999; PubMed Scopus Google Scholar). The DNA to the of the complexes with the carboxyl-terminal domain was using a DNA of the the DNA was as to the component of the at in the as described in (35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In of complexes of different of protein to DNA and The protein were of the the that the amide I was not by DNA changes by interaction with the the of the thermal stability of the a was the and the was on a were by from to °C at a rate of were using a of temperature were and the with a J.L.R. I. I. M.A. J. PubMed Scopus Google Scholar). The was as described J.L.R. J. J.M. Biochemistry. 1994; PubMed Scopus Google Scholar). were in The DNA to the at temperature was using a DNA at the temperature and concentration. The was using the at as described In the amide the of and DNA were similar not to the protein and IR of the Carboxyl-terminal of Histone H1 in and in have used IR spectroscopy to the secondary structure of the carboxyl-terminal domains of the histone H1 subtypes H10 (C-H10) and H1t in aqueous and in to and are in I of the carboxyl-terminal domain of histone The shows the of the C-H10, and the shows the of the C-H1t. The were in and in solution at The was mm mm The protein was of secondary structure of the carboxyl-terminal domain of histones H10 and H1t in aqueous and and of the components of the amide I of the C-H10 and C-H1t in aqueous and The were to the of structure by from the at as described and TFE, and of the components of the amide I of the C-H10 and C-H1t in aqueous and The were to the of structure by from the at as described and in a new In aqueous solution, the amide of C-H10 and C-H1t was by the random at with of the amide The other component at with of the in C-H10 and in C-H1t, was to turns. In studies with H1 peptides, the component at was to the in rapid equilibrium with the unfolded that in the presence of secondary structure stabilizers are transformed into helical structure (35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, R. Ponte I. Collado M. Arrondo J.L.R. Jiménez M.A. Rico M. Suau P. J. Biol. Chem. 2001; 276: 46429-46435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, J. M. R.A. R.A. J. Mol. Biol. 1988; PubMed Scopus Google Scholar). of at and structure were also In TFE, C-H10 and C-H1t were extensively α-helix and were with a component of random that are not in The of at in C-H10 and in C-H1t. In to turns, component could contain a proportion of the 310 helix M. C. Chapman D. Biochemistry. PubMed Scopus Google Scholar, A.P. Nature. 1992; PubMed Scopus Google Scholar, S. Biochemistry. 1996; PubMed Scopus Google Scholar), in which the proportion of helical structure would The of α-helix by from the at Biochemistry. PubMed Scopus Google Scholar) were similar to by IR spectroscopy for C-H10 and C-H1t IR of the Carboxyl-terminal of Histone H1 to have the secondary structure of the carboxyl-terminal domain of histone H1 bound to DNA. The of the complexes were in and to the of the amide I has a on the of some components of the amide are or not at J.L.R. Biophys. Mol. Biol. 1999; PubMed Scopus Google Scholar). The of and the α-helix are by and are thus at similar in and the α-helix at and at In has a on the of the random that in it with the α-helix, in it with loops. the α-helix could be in could be in α-helix and are present in the protein structure, the of random be either from the of the components in and or from the of the components in and as by the expression in is the of the amide I component in either and is the of random the of the different secondary structure elements in and be the of the of random from the of the components either in and or in and the of the amide I components of α-helix, loops, and random components at the in and turn components in are to The complexes of the carboxyl-terminal domain with DNA were in and mm In mm the of the complexes of C-H10 with DNA in and turns, α-helix, 25% open loops, β-structure, and 7% random The amount of random was from the of the components in and and from the of the components in and The were and The representation of the different of secondary structure in C-H1t bound to DNA was similar to that of C-H10, although some were a higher amount of random a amount of α-helix and open and a higher amount of and and I of the of the C-H1t bound to DNA. The shows the in and the shows in The salt or mm is The DNA to the of the complexes was as described and of secondary structure of the carboxyl-terminal domain of histones H10 and H1t bound to with mm mm to the the components at in and to the the components at in to the the components at in and to the the components at in The to the the components at in and The to the the components at in in a new the salt was to mm the DNA-bound carboxyl-terminal domains of H10 and H1t significant structural In the the protein fully as by the of a random In C-H10, the of the random was with an of α-helix, β-structure, and turns, which of and in the of open from 25% in mm to in mm was also The of was most for C-H1t, the α-helix also to the of the structural motifs not and in solution, the component at could contain a proportion of the 310 helix. the changes in the amide components of the DNA-bound C-H1t upon in the 20–80 °C The DNA to the at temperature was using a DNA at the temperature and the was using the at as described and free DNA DNA, the of and DNA were similar not to the amide of the were in either or mm at a of In mm the α-helix and components to °C and they to a residual which may at higher with the of the α-helix and the the random component from to The low a temperature interval with a of an in was with the of the low and In mm the structure of the DNA-bound protein was more the of α-helix, and random to of the low and a of the turn component was and We have the secondary structure of the carboxyl-terminal domain of linker histone subtypes H10 and H1t free in solution and bound to DNA. The of the entire carboxyl-terminal domain is important in the study of that are not to specific subdomain (27Lu X. Hansen J.C. J. Biol. Chem. 2004; 279: 8701-8707Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) but are determined by the domain, such as affinity (19Th'ng J.P.H. Sung R. Ye M. Hendzel M.J. J. Biol. Chem. 2005; 280: 27809-27814Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) or apoptotic nuclease (31Widlak P. Kalinowska M. Parseghian M.H. Lu X. Hansen J.C. Garrard W.T. Biochemistry. 2005; 44: 7871-7878Crossref PubMed Scopus (54) Google Scholar). The IR of the carboxyl-terminal domain in aqueous solution were by the random with of the amide I However, in most highly unfolded an in C-H10 and in at was the presence of turn-like conformations in rapid equilibrium with (35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, R. Ponte I. Collado M. Arrondo J.L.R. Jiménez M.A. Rico M. Suau P. J. Biol. Chem. 2001; 276: 46429-46435Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). of structure consisting of at and were also present in the free interaction with DNA, the carboxyl-terminal domain an extensively folded The structure of the bound domain showed a significant dependence on salt concentration. In mm the DNA-bound C-H10 contained α-helix and open with a residual 7% random In physiological salt (140 the carboxyl terminus fully as by the of random Under these conditions, C-H10 contained 24% α-helix, 25% β-structure, open loops, and turns. The component at in and in the complexes with DNA could or in part from the 310 helix. This has been to the 310 helix in studies M. C. Chapman D. Biochemistry. PubMed Scopus Google Scholar, S. Biochemistry. 1996; PubMed Scopus Google Scholar). The presence of the 310 helix was demonstrated in a peptide from the H10 carboxyl-terminal domain (34Vila R. Ponte I. Jiménez M.A. Rico M. Suau P. Protein Sci. 2000; 9: 1-10PubMed Google Scholar, 35Vila R. Ponte I. Collado M. Arrondo J.L.R. Suau P. J. Biol. Chem. 2001; 276: 30898-30903Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The amount of α-helix thus be as a of the amount of helical structure. All of the structural motifs in the carboxyl-terminal domain have the to with the or DNA Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). The dependence of the secondary structure of the DNA-bound carboxyl-terminal domain on salt although is an of which could have a role in chromatin structure and Despite the sequence C-H10 and C-H1t sequence the to have secondary structure in mm and mm This that may to the secondary structure of the DNA-bound domain in different subtypes. The secondary structure of the DNA-bound domain appears to be extremely stable. In mm the α-helix and the to at the low with a of In the α-helix and the are more and changes are in their to the temperature of The low in a temperature interval similar to that at low with a in the proportion of turns. The thermal stability of the DNA-bound may from of the charge of the protein by the DNA the proportion of residues in the carboxyl terminus charge have a on the for charge in complexes is by the of in complexes (30Roque A. Orrego M. Ponte I. Suau P. Nucleic Acids Res. 2004; 32: 6111-6119Crossref PubMed Scopus (31) Google Scholar, L. Suau P. Biophys. Chem. PubMed Scopus (15) Google Scholar). In complexes the of complexes by condensation the DNA charge is fully charge L. Suau P. Biophys. Chem. PubMed Scopus (15) Google Scholar, Biopolymers. PubMed Scopus Google Scholar, R.W. Biochemistry. PubMed Scopus Google Scholar). could contribute to in a more specific In the helical folding to a because of the of the helix with of the residues on of the helix and of the residues on the other (34Vila R. Ponte I. Jiménez M.A. Rico M. Suau P. Protein Sci. 2000; 9: 1-10PubMed Google Scholar). The carboxyl-terminal domain appears to belong to the of also as unfolded or C. Curr. Biol. 2004; PubMed Scopus Google Scholar, Curr. Biol. 2005; 15: PubMed Scopus Google Scholar, 2005; Scopus Google Scholar). In free solution, these specific secondary or structure and are of an of disordered into stable secondary or structures upon binding to their such as other proteins, acids, or is characterized by a low sequence a low of amino acids, and a of and amino All of these are present in the carboxyl-terminal domains and also in the amino-terminal domains of H1 I. R. Suau P. Mol. Biol. Evol. PubMed Scopus (33) Google Scholar). In disordered regions are present in by which is also the with H1 P. Nucleic Acids Res. 2004; 32: PubMed Scopus Google Scholar). The H1 globular domain is to be in specific binding in folded carboxyl-terminal domain with charge could with binding that the folding of the carboxyl terminus is with charge binding to such as DNA, that is already in with histones may to folding and affinity of the carboxyl of the of would thus be to the of specific H1 binding sites in