J. Mol. Biol. (1990) 212, 253-257

Crystallization V. Grazianol,

of the Globular

Domain of Histone H5

S. E. Gerchman’, A. J. Wonacott’Jf, R. M. SweetI, J. R. E. Wells2 S. W. White1 and V. Ramakrishnanl ‘Biology Department Brookhaven National Laboratory Upton, NY 11973, U.S.A. 2Department of Biochemistry University of Adelaide Adelaide, South Australia 5001, Australia (Received 2 November 1989)

The globular domain of histone Hl/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene t,hat codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 a (1 L%= @l nm) resolution. The crystals are orthorhombic with unit cell dimensions of a=80.1 A,b=67.5Aandc=3808.

particle. Thus the presence of Hl/H5 on the nucleosome protects 10 base-pairs at each end of the nucleosomal DNA. Allan et al. (1980) showed that the globular domain of Hl/H5 by itself can bind to the nucleosome and protect nucleosomal DNA from digestion. High-resolution structures of the globular domains of HI and H5 will be essential to understand the interaction of these proteins with the nucleosome. The tertiary fold of the globular domain of H5 has been detemined by two-dimensional nuclear magnetic resonance (Clore et al.. 1987). It’ would be useful to complement this information with a high-

The linker histones Hl and H5 bind to the nucleosome and are crucial for the formation of the 30 nm filament, which is the predominant form of chromatin higher order structure in eukaryotic cells (for a review see McGhee & Felsenfeld, 1980; Thomas, 1984; Wu et al., 1986). In addition, a recent report demonstrates that Hl is a recombinase that catalyzes ATP-independent strand transfer (Kawasaki et al., 1989). Histones Hl and H5 are homologous (Yaguchi et al., 1979; Briand et al., 1980). Both histones consist of disordered N- and C-terminal domains t.hat flank a central: trypsin-resistant, globular domain (Hartman et a,Z., 1977; Aviles et aE., 1978). In H5, t,he globular domain produced by trypsin digestion spans residues 21 to 100. The C-terminal domain is extremely basic: almost half of it consists of lysines and arginines. H5 is a variant of Hl found in avian erythrocytes. It is more basic than Hl as a result of several lysine to arginine substitutions, and binds more tightly to chromatin. Because the accumulation of H5 is correlated with the maturation of the erthrocyte, the protein may be associated with a reduction in gene expression (Weintraub et aE., 1971). Digestion of chromatin by micrococcal nuclease results in the chromatosome (Simpson, 1978), which is a nucleosome consisting of approximately 165 base-pairs of DNA surrounding the histone octamer, and one molecule of Hl/H5. Removal of HI/H5 results in an extra 20 base-pairs of nucleosomal DNA being digested to produce the nucleosome core t Permanent address: Blackett C!ollege, London SW? 2BZ1 UK.

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Figure 1. Plasmid containing the coding sequence for the globular domain of H5. The plasmid is based on the expression vector pet 3c (Rosenberg el al., 1987), that uses the bacteriophage T7 promoter and transcription signals, and the T7 gene 10 ribosome initiation signal.

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Figure 2. Expression of GH5 in E. coli using the ‘I’7 expression system (see Fig. I ). ‘I’hr figure shous itn SDS. polyacrylamide gel of total cell protein at times 0, 15. 30, 60 and 120 min after induction. The first lane shows markers. with the numbers indicating the molecular weights in kl)a. resolution crystal structure that~ accurately determined the positions of the amino acid side-chains. Despite the characterization of the globular domain over a decade ago, there have been no reports of its crystallization. It is possible that the fragment’ produced by trypsin digestion is heterogeneous,

owing to the presence of c*losel\: spaced t.rypsin cleavage sites on Hl/H5. This heterogeneity may have prevented t,he information of well-ordered crystals. It is also possible that post-translational modifications may contribute to addition hetcrogeneity. These difficulties can br overcome I)>

Figure 3. Protection of nucleosomal DNA by GH5 produced in E. coli. Chromatin was stripped of Hl/H5, reconstituted with GH5, and digested with micrococcal nuclease as described previously (Allan et al., 1980; Graziano et al., 1988). The digestion times were 0, 4, 8, 16 and 32 min. The gel shows DNA from the digestions run on a 6% (w/v) acrylamide gel. Lane 1, T7 Hoe11 digest as molecular weight markers, with the sizes in base-pairs indicated for 2 of the bands; lanes 2 to 6, native chromatin; lanes 8 to 12 stripped chromatin; lanes 14 to 18, stripped chromatin reconstituted with GH5. Lanes 7, 13 and 19 show DNA from nucleosome core particles run as markers. Native and reconstituted chromatin show bands, corresponding to the chromatosome (165 bp) and the nucleosome core particle (146 bp), respectively. The 165 bp band arises from the protection of nucloosomal DNA by H5 or GH5. Stripped chromatin, on the other hand, is progressively digested to produce nucleosome core particles. bp, base-pairs.

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V. Craziano

expressing in Escherichia coli the portion of the H5 gene that codes for the globular domain. A clone containing the chicken H5 gene (Krieg et al., 1983) was used to construct a gene coding for the H5 globular fragment as follows. The coding sequence for H5 was introduced into the expression vector pet-3c, which contains a strong T7 promoter and the T7 gene 10 ribosome binding site (Rosenberg et al, 1987). A stop codon was introduced at the SmaI site at position 318. In addition, bases 10 to 66 (inclusive) were deleted. The resulting gene codes for a protein that consists of the first three residues of H5 followed by residues 23 to 106. Thus the product corresponds to a fragment, GH5, that is slightly larger than the fragment produced by trypsin digestion. We found it necessary to include the first nine bases of the H5 gene for efficient protein synthesis on induction, suggesting t’hat some of these bases may be involved in the binding of the ribosome to the initiation site. The resulting plasmid, pVG13, is shown in Figure 1. The plasmid was used to transform the E. coli strain BL21(DE3), which contains the T7 RNA polymerase gene on the host chromosome under control of the lac promoter. Figure 2 shows an SDS/polyacrvlamide gel of total cell protein sampled at various times after induction of the cells with isopropyl-fl-n-thiogalactoside. GH5 was purified from cell lysates by ion-exchange chromatography on a Whatman CM-52 column at pH 50, followed by chromatography on Pharmacia FPLC Mono-Q column at pH 8.0. A one-liter growth typically yielded 10 to 15 mg of pure GH5. The protein was tested for its ability to bind to the nucleosome by the methods of Allan et al. (1980). Figure 3 shows a gel of chromatin reconstituted with GH5 digested for various times with micrococcal nuclease. The 165 base-pair product observed for native and reconstituted chromatin is evidence of the protection of nucleosomal DNA by GH5. Crystallization trials were set up by the hanging drop method. Single crystals of GH5 were obtained at 4°C in two forms. The first, which was obtained in 3.5 M-(NH,),SO, (pH 80) did not diffract beyond 6 A resolution (1 A = 61 nm). The second form; which was obtained in 2.2 M-phosphate (pH 8.2) diffracted to beyond 2.5 A resolution, and was chosen for further analysis. The largest crystals obtained so far were rectangular plates with dimensions of about 0.1 mm x 62 mm x 0.2 mm. Crystals were examined on beam-line X-12C at the National Synchrotron Light Source at Brookhaven National Laboratory. Initially, an oscillation camera was used to do a 5” rotation about the hlc0 zone, to determine approximate unit cell parameters and look for systematic absences. Subsequently, limited three-dimensional data were collected with the Enraf-Nonius FAST area the MADNES program using detector, (Messerschmidt & Pflugrath, 1987). Auto-indexing and refinement yielded cell parameters that agreed well with those determined from film measurements. At 4°C. the GH5 crystals grown in phosphate

et al.

cell dimensions were orthorhombic, with 01 a = SO.1 A, h = 67.5 8. c = 38.0 Ak. Upon warming t’o room temperat,ure (approx. 20°C). the cryst.als went t,hrough a disordered phase (which was observed on the area detector as a loss of the higherorder reflections and a smearing of the low order reflections) and finally equilibrated to an ordered structure in which the new dimensions were a = 79.1 A, 6 = 61.3 A, c = 36.8 A. Despite the change of 6.2 A in the b axis, and a shrinkage in volume of 150/o, the quality of the diffraction was unaffected: the crystals at room temperature also diffracted to beyond 2.5 A resolution. Examination of low order reflections along the hO0 and O/?$directions indicated systematic absences consistent wit,h the space groups P2,2,2, or P2,2,2. Assuming two molecules per asymmetric unit, this leads to a volume of 2.68 A3/Dalton for the crystals at 4°C and 2.32 A3/Dalton at room temperature. While it appears that temperature will need to be controlled to obtain reproducible cell dimensions. these crystals are suitable to begin a high resolution structure determination of GH5. Since the protein has been obtained from E. coli, it should be possible to produce it in selenated form. by growth in a medium containing selenomethionine. GH5 contains a single internal methionine and a possible N-terminal methionine. Phases could then be obtained by anomalous scattering from the selenium atom, using the multiwavelength anomalous dispersion technique (Hendrickson et al., 1989). Similar attempts to crystallize a genetically engineered globular fragment of Hl are also underway in our laboratory. We thank Drs J. J. Dunn and F. W. Studier for their help and advice with cloning procedures using the ‘I’7 system. This work was supported by grant GM 42796 from the NIH and by the Office of Health and Environmental Research of the United States Department of Energy.

References Allan, J.. Hartman, P. G.. Crane-Robinson, D. 62.Aviles. F. X. (1980) Nature (London), 288, 67.5-679. Aviles, F. J., Chapman, G. E., Kneale, G. G., C%xneRobinson, C. & Bradbury. E. M. (1978). Ew. J. Biochem. 88, 363-371.. Briand, G., Kmiecik, D., Sautiere, I’.. Wouters, D.. Boric Loy, O., Biserte, G., Masen, A. & Champagne, M. (1980). FEBS. Letters, 112, 1477151. Clore, G. M., Gronenborn. A. M.. Nilges, M., Sukumaran, I). K & Zarbock, ,J. (1987). EMBO .I. 6, 1833-1X42. Graziano. V., Gerchman. S. E. & Ramakrishnan. I’. (1988). .I. Mol. Riol. 203. 997-1007. Hartman. 1’. G.. Chapman. Cr. E., .Moss. ‘I’. C%Hraclbury 1 E. M. (1977). Eur. .I. Biochrrtr. 77, 45 51. Henrickson. )V. :I.. Horton. .J. R.. Krishna .\lurth~. H. 31.. Pahhr. :I. & Smith. .J.T,. (lQ8Q). Itr Synch~rotron Ra&fion i?l AYructurr11 ISiolog!y (SwvHt. R. M. 8 LVoodhead. A. D.. t~ls), pp. 31 ii--344. I’lenum Press. New York. Kawasaki. 1.. Sugano. S. 6t Ike&. H. (lQ8Q). /‘rf~c, .VII/. Acad. SC

Crystallization of the globular domain of histone H5.

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed i...
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