Biochemistry zyxwvut 1993,32, zyxwvu 11345-1 1351 11345 Homo- and Heteronuclear Two-Dimensional NMR Studies of the Globular Domain of Histone H 1 zyxwv : Sequential Assignment and Secondary Structure7 Corinne Cerf,',$ Guy Lippens,$ Serge Muyldermans,s Alain Segers,i zyxwv V. Ramakrishnan,ll Shoshana J. Wodak,t Klaas Hallenga,l and Lode Wynd Unitk de Conformation des Macromolbcules Biologiques (UCMB), CP 16011 6, Universitk Libre de Bruxelles, Avenue P. Hbger, B- 1050 Brussels, Belgium, Instituut voor Molekulaire Biologie, Vrije Universiteit Brussel, Paardenstraat 65, B- 1640 Sint-Genesius-Rode, Belgium, Biology Department, Brookhaven National Laboratory, Upton, New York 11973, and Corvas Int. N. V. at zyxwvuts UCMB and Corvas Int. N. V., Plateaustraat 22, B-9000 Ghent, Belgium Received July 26, zyxwvu 1993' ABSTRACT: A recombinant 75 amino acid polypeptide corresponding to the globular domain of the chicken histone H1 (GH1) has been studied by 'H homonuclear and lH-15N heteronuclear 2D NMR spectroscopy. Sequential assignment of the backbone and 8-proton resonances has enabled us to determine the secondary structure of GH1. It was found to consist of three helical regions (T7S17, L25-Y37, E40-K56) and probably a @-hairpin (L59-L73). This structure is similar to the structure of the globular domain of histone H5 (GH5) obtained both by NMR spectroscopy [Zarbock et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7628-7632; Clore et al. (1987) EMBO J. 6, 1833-18421 and by X-ray crystallography [Ramakrishnan et al. (1993) Nature 362,219-2231. The 8-hairpin as suggested for GH1 is also present in the X-ray structure of GH5 but has not been reported for the NMR structure of GHS. The linker histone H1 is a component of the nucleosome, and is responsiblefor the foldingand maintenanceof chromatin into the 30-nmfilament (Thoma et al., 1979). It is also thought to play a direct role in the regulation of DNA transcription and replication, and in cell proliferation (Croston et al., 1991; Zlatanova, 1990; Kamakaka & Thomas, 1990; Sun et al., 1989). The histones H1 constitute a family with many variants, differing in their affinity for chromatin (Kumar & Walker, 1980). Several variants are simultaneously present in a single cell. For example, the nucleated erythrocytes of birds contain both H1 and H5, the latter being an extreme variant of H1. H 1and its variants have a multidomain structure (Bradbury et al., 1975). There is a central, trypsin-resistant, globular domain (GHl),' flanked by extended N- and C-terminal domains with a high content of lysines and arginines and thus very positively charged. GH1 is essential for the binding of H1 to the nucleosome. It is the part of H1 which is responsible for the specific positioning of the protein on the nucleosome, and it confers the same nucleosome protection from micro- C.C. is a Research Assistant of the National Fund for Scientific Research (Belgium). V.R. is supported by NIH Grant GM42796 and by the Office of Health and Environmental Research of the U.S. Department of Energy. Part of this work was financially supported by the National Fund for ScientificResearch (Belgium). This paper presents research results of the Belgian Programme on Interuniversity Poles of Attractioninitiated by the Belgian State, Prime Minister's Office, Science Policy Programming. * To whom correspondenceshould be addressed. zyxwvutsrq t Universitd Libre de Bruxelles. zyxwvutsrq 4 Vrije Universiteit Brussel. 11 Brookhaven National Laboratory. zyxwvutsr I Corvas Int. N. V. * Abstract published in Advance ACS Abstracts, October 1, 1993. Abbreviations: GH1, globulardomain of histone H1; GH5, globular domain of histone H5; 1 D, one-dimensional; 2D, two-dimensional; NMR, nuclear magnetic resonance; DQF-COSY, double-quantum-filtered J-correlated spectroscopy; DQSY, double-quantum spectroscopy; TOC- SY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear singlequantum coherence; HMQC, heteronuclear multiple-quantum coherence; SCUBA, stimulated cross-peaks under bleached alphas. 0006-2960/93/0432-11345%04.00/0 coccal nuclease digestion as complete H1 (Allan et al., 1980; Buckle et al., 1992). The isolated globulardomains of different H 1 variants also show different affinities for chromatin (Thoma et al., 1983). Natural GH 1, obtained by trypsin digestion of complete chicken histone H 1, turned out to be too heterogeneous to allow successful NMR studies. The breakthrough was achieved by working with a 75-residue recombinant polypep- tide having the sequence of a particular GH1 variant. As such, a more homogeneous preparation could be obtained compared to the natural fragment. This recombinant GH1 was studied by means of lH homonuclear and lH-lSN heteronuclear 2D NMR spectroscopy. Comparison between analyzable parts of the natural GH1 spectra and the corre- sponding parts of the recombinant GH1 spectra permitted us to verify that both polypeptides possess the same fold (Segers, 1991). Here we present the assignment of 'H and 15N backbone resonances (up to @-protons)and the secondary structure of GH1. These data are discussed in the light of GH5 structures obtained previously by NMR (Clore et al., 1987) and more recently by X-ray crystallography (Ra- makrishnan et al., 1993). MATERIALS AND METHODS Preparation of Natural CHI. Natural chicken erythrocyte GH1 was prepared and purified on a TSK SP-5PW Ultropac (LKB) ion-exchange column accordingto Segers et al. (1991). Preparation of Recombinant C H I . A GH1 gene was synthesized on the basis of the amino acid sequence of the globular domain of the 11L H1 variant from chicken erythrocyte (methionine residue +40-113 fragment). It is noticeable that this sequence is shared by globular domains of other chicken H1 variants (36-109 segment of .10 H1, 37-110 segment of 11R H1) (Coles et al., 1987). The gene was cloned in a PET 3c-based vector (Studier et al., 1990) and expressed in the Escherichia coli BLZl(DE3) cells. Cells were grown until late-logarithmic phase in LB medium containing ampicillin (1 00 pg/mL), or in minimal medium 0 1993 American Chemical Society