close

Вход

Забыли?

вход по аккаунту

?

642

код для вставкиСкачать
PROTEINS: Structure, Function, and Genetics 24:269-271 (1996)
Crystallization and Preliminary X-Ray
Characterization of the Methanothermus fervidus
Histones HMfA and HMfB
Klaas Decanniere,' Kathleen Sandman: John N. Reeve: and Udo Heinemann'
'Forschungsgruppe Kristallographie, Max-Delbruck-Centrumfur Molekulare Medizin, 0-13122 Berlin, Germany;
and 2Department of Microbiology, The Ohio State University, Columbus, Ohio 43210
HMfA and HMfB are histone
ABSTRACT
proteins from the thermophilic archaeon Methanothermus fervidus. They wrap DNA into
nucleosome-like structures and appear to represent the basic core histone fold. HMfA was
crystallized in space groups P4,2,2 and
P2,2,2,. HMfB crystallized in space group
P2,2,2, while a selenomethionine-substituted
variant, SeMet-HMfB,yielded crystals in C222,.
In all crystal forms HMfA, HMfB, or SeMetHMfB may be present as homodimers.
0 1996 Wiley-Liss, Inc.
Key words: Archaea, hyperthermophiles, histone fold, X-ray diffraction, anomalous dispersion
INTRODUCTION
Methanothermus fervidus is a methanogenic archaeon that grows optimally a t 83°C. It contains two
small proteins, HMfA and HMfB, which bind double-stranded DNA in ~ i t r o , ' - ~forming structures
that resemble eukaryal nucleosomes. They bind to
DNA as dimers or tetramers, and both homodimers
and HMfA-HMfB heterodimers have been observed
to exist in vivo5 The amino acid sequences of HMfA
and HMfB differ at only 12 of 68 or 69 positions,
respectively. Sequence alignments reveal homologies between HMfMHMfB and the eukaryal core
histones HZA, H2B, H3, and H4.l The closest match
is observed between HMfB and the consensus sequence for the C-terminal part of H4,, which share
29% of identical and 55% of conserved residues. The
similarity to histone H4 is particularly high in three
regions that adopt a n a-helical conformation and
form the histone-fold motif within the structured
core of the histone ~ c t a m e r .HMfA
~
and HMfB
therefore appear to represent the basic core histone
motif.' Here we report the crystallization and preliminary crystal data for HMfA and HMfB. A variant of HMfB in which residues Met-1 and Met-35
have been replaced by selenomethionine residues
(SeMet-HMfB) has also been produced and crystallized.
0 1996 WILEY-LISS, INC.
MATERIALS AND METHODS
Generation of Selenomethionine
Variant, SeMet-HMfB
E . coli JM105 was made awotrophic for methionine by P1 transduction of metC162::TnlO (tet')
from E . coli NK6027 (obtained from the E . coli Genetic Stock Center), and the resulting strain was
transformed with the hmfB expression vector
pKS323
All cultures were grown with 20
pg/ml ampicillin and 10 pg/ml tetracycline. An
overnight culture of E . coli JM105-metC162::TnlO
(pKS323) was grown in M9 medium" supplemented
with 5 pg methionine/ml, and a 100 ml starter culture was grown in M9 containing 5% LB and 50 pg
D,L-selenomethionine/ml (Sigma, St. Louis, MO) by
inoculation with a 100-fold dilution of the overnight
culture. After 5 h growth at 37°C the starter culture
(OD,,, of 0.3) was diluted into a 10 L fermentor
containing M9 and 50 pg selenomethionine/ml.
When the culture reached a n OD,,, of 0.2 (-17 h
later), isoptopyl P-D-thiogalacto pyranoside (IPTG)
was added to 0.4 mM to induce the synthesis of
SeMet-HMfB. The culture was harvested 6 h later.
The total yields from two 20 L cultures were 11.5 g
of E. coli cells (wet weight) and 5 mg of purified
SeMet-HMfB.
Protein Purification and Characterization
Cell growth and purification of HMfA was as described," except that the growth medium was Superbroth." Recombinant HMfB (rHMfB) was purified as describedg except that the heparin sepharose
column was equilibrated with 30 mM potassium citrate, 50 mM Tris, pH 8.0, and the rHMfB was
eluted with a 30-200 mM gradient of potassium citrate in 50 mM Tris, pH 8.0. Amino acid compositions were determined directly for rHMfB and
SeMet-HMfB. In SeMet-HMfB > 99% of the methionyl residues in rHMfB was replaced by selenomethionyl residues.
Received June 22, 1995; revision accepted August 28, 1995.
Address reprint requests to Udo Heinernann, Forschungsgruppe Kristallographie, Max-Delbruck-Centrum fur Molekulare Medizin, Robert-RGssle-Str. 10, D-13122 Berlin, Germany.
270
K. DECANNIERE ET AL.
TABLE I. Crystallization Conditions
Reservoir
Crystallization
time (days)
Max. crystal
size (mm)
HMfA tetragonal
0.1 M Na-acetate,
pH 4.6, 2.2 M NaCl
HMfA orthorhombic
2.6 M (NH,)2S04
HMfB
0.15 M cacodylate, pH 6.5,
0.3 M Zn-acetate, 17%
(w/v) PEG 8000
SeMet-HMfB
0.1 M MES pH 6.5,
0.2 M (NH4),S0,,
30% (wiv) PEG
monomethyl
ether 5000
2-3
1-2
1-2
7
0.4
X
0.4
0.6
X
0.2 x 0.2 x 0.4
0.3
X
0.5
X
0.7
0.15 x 0.3 x 0.5
TABLE 11. Crystallographic Characterization
Space group
a (A)
b (A)
c (A)
VM (A3/Da)
Protein monomers per asymmetric unit*
HMfA tetragonal
P4,2,2
41.24
41.24
81.19
2.26
1
HMfA orthorhombic
p212121
33.68
52.65
67.22
1.95
2
HMfB
P2,2,2
31.24
39.41
61.47
2.47
1
SeMet-HMfB
(2222,
29.88
59.94
75.50
2.21
1
*Chosen such as to yield reasonable V, values.
Growth and Preliminary
Characterization of Crystals
All crystallization experiments used the hanging
drop vapor diffusion method at room temperature.
HMfA and HMfB were concentrated to 10-20 mglml
in their buffer solutions (approximately 100 mM potassium citrate, 50 mM Tris-HC1, pH 8.0). Initial
crystallization conditions were established by mixing 1 pl of protein solution with 1 pl of precipitant
solution and equilibration with the precipitant
solution in an extended sparse matrix screen.l3,l4
For X-ray diffraction experiments, crystals were
mounted in sealed glass capillaries. In most cases
the crystals proved sensitive to changes of their
mother liquor and were thus mounted straight from
the drop. Diffraction data sets were collected on 180
mm or 300 mm MarResearch (Hamburg, Germany)
imaging plate systems. For in-house experiments,
CuK, radiation was produced by Rigaku-Denki (Tokyo, Japan) RU H2R direct-drive rotating-anode
generators. For use with the small imaging plate,
the generator was operated at 44 kV, 110 mA with
0.3 mm focal spot size and graphite monochromator.
For use with the large plate, operation conditions
were 44 kV, 68 mA with 0.2 mm focus, monochromatization by Ni-coated double-focusing mirrors
(Charles Supper, Natick, MA) and filtering through
a 17.5 pm Ni foil. Some data sets were collected at
beamlines X31 or BW6 of the EMBL outstation a t
the Deutsche Elektronensynchrotron (DESY, Hamburg). Primary data were processed with DENZ015
and scaled with the programs SCALA and AGROVATA of the CCP4 program package.16 The Laue
symmetry and screw axes were verified by precession photography.
RESULTS AND DISCUSSION
After refinement of the crystallization conditions
found by the initial screen, proteins HMfA, HMfB,
and SeMet-HMfB formed single crystals suitable for
high-resolution structure analysis (Table I). Crystallographic parameters for the M. fervidus histones
are summarized in Table 11. The two different crystallization conditions for HMfA give rise to tetragonal and orthorhombic crystal forms. Unfortunately,
SeMet-HMfB does not crystallize isomorphously
with HMfB. However, the crystal packing may be
similar, since space group P2,2,2 of HMf33 is a subgroup of C222, found for SeMet-HMfB, and lattice
parameters a, b, c of the C222, cell are close to a', c',
2b' of the P2,2,2 cell, respectively. Alternatively, c
may correspond to the b'ic' diagonal. The packing
parameters V , are in the expected range,17 between
1.95and 2.47 A31Da, if a protein dimer is assumed to
occupy the asymmetric unit in orthorhombic HMfA
and a monomer i n all other crystals. We note that
symmetric dimers with crystallographic dyad axes
may be present in all crystal forms in which the
asymmetric unit contains a protein monomer.
X-Ray diffraction data sets were collected either using in-house facilities or at DESY, Hamburg (Table
111).Completeness and Rmergevalues (Rmerge= ZlZ,,
12&, where Zi,jare the measurements
contributing to the mean reflection intensity, <Ii>>,
for the entire data sets and for the highest shells,
indicate reasonable data up to the diffraction limits given. It is hoped that anomalous dispersion
measurements using the selenium atoms of
SeMet-HMfB will help solve the phase problem
for the structure analysis by established procedures.18,19
271
CRYSTALLIZATION OF ARCHAEAL HISTONES
TABLE 111. X-Ray Diffraction Data
X-ray source
Wavelength (A)
Detector
Whole data set
Resolution limit (A)
Completeness (%)
HMfA tetragonal
RU H2B
HMfA orthorhombic
DESY, BW6
HMfB
RU H2B
SeMet-HMfB
DESY, X31
1.5418
IP 180 mm
1.030
IP 180 mm
1.5418
IP 300 mm
0.877
IP 180 mm
2.1
84.6
7.4
1.9
90.4
6.0
1.9
96.5
5.3
2.1
97.2
8.6
2.15-2.10
59.4
23.7
1.96-1.90
85.4
19.3
1.97-1.90
97.2
19.6
2.17-2.10
90.2
15.8
Rrnerge
Highest shell
Resolution limits (A)
Completeness (%)
Rmerze (%)
ACKNOWLEDGMENTS
X-Ray diffraction experiments were in part performed a t the EMBL Outstation at DESY, Hamburg, with kind help from G. Evans. The help of J.J.
Muller and A. Knespel in setting up the X-ray laboratory at the MDC is gratefully acknowledged. We
thank Don Ordaz, OSU Fermentation Facility, John
Lowbridge, OSU Biochemical Instrumentation Center, and A. Rampersaud for gift of P1 lysates and
protocols. This work was supported by grants from
the Deutsche Forschungsgemeinschaft under SFB
344JYE7 and from the Fonds der Chemischen Industrie to U.H. and from the Office of Naval Research
(N00014-92-5-1932)to J.N.R.
REFERENCES
1. Sandman, K., Krzycki, J.A., Dobrinski, B., Lurz, R.,
Reeve, J.N. HMf, a DNA-binding protein isolated from the
hvDerthermoDhilic archaeon Methanothermus feruidus. is
mist closely ;elated to histones. Proc. Natl. Acid. Sci. USA
87:5788-5791,1990,
2. Musgrave, D.R., Sandman, K.M., Reeve, J.N. DNA bindine bv the archaeal histone HMf results in uositive supercoaiig. Roc. Natl. Acad. Sci. USA 88:1039?-10401,1991.
3. Howard, M.T., Sandman, K., Reeve, J.N., Grifith, J.D.
HMf, a histone-related protein from the hyperthermophilic
archaeon Methanothermus feruidus. binds preferentially
to DNA containing phased tracts of adenines. J . Bacteriol.
174:7864-7867, 1992.
4. Stroup, D., Reeve, J.N. Histone HMf from the hyperthermophilic archaeon Methanothermus feruidus binds to DNA
in vitro using physiological conditions. FEMS Microbiol.
Letters 91:271-276, 1992.
5. Grayling, R.A., Becktel, W.J., Reeve, J.N. Structure and
stability of histone HMf from the hyperthermophilic archaeon Methanothermus feruidus. Biochemistry 34:84418448,1995.
6. Wells. D.. McBride. C. A comurehensive comuilation and
alignment of histones and h&tone genes. N h e i c Acids
Res. 17(suppl.):r311-r346, 1989.
7. Arents, G., Burlingame, R.W., Wang, B.-C., Love, W.E.,
Moudrianakis, E.N. The nucleosomal core histone octamer
at 3.1 b resolution: A tripartite protein assembly and a
left-handed superhelix. Proc. Natl. Acad. Sci. USA 88:
10148-10152, 1991.
8. Arents, G., Moudrianakis, E.N. Topography of the histone
octamer surface: Repeating structural motifs utilized in
the docking of nucleosomal DNA. Proc. Natl. Acad. Sci.
USA 90:10489-10493, 1993.
9. Sandman, K., Grayling, R.A., Dobrinski, B., Lurz, R.,
Reeve, J.N. Growth phase dependent synthesis of histones
in the archaeon Methanotherrnus feruidus. Proc. Natl.
Acad. Sci. USA 91:12624-12628, 1994.
10. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. Short Protocols in Molecular Biology. New York John Wiley & Sons, 1989.
11. Sandman, K., Grayling, R.A., Reeve, J.N. Improved N-terminal processing of recombinant proteins synthesized in
Escherzchia coli. Bio/Technology 13504-506, 1995.
12. Kuo, C.-F., McRee, D.E., Cunningham, R.P., Tainer, J.A.
Purification, crystallization and space group determination of DNA repair enzyme exonuclease I11 from E. coli. J .
Mol. Biol. 229:239-242, 1993.
13. Jancarik, J., Kim, S.-H. Sparse matrix sampling: A screening method for crystallization of proteins. J . Appl. Crystallogr. 24:409-411, 1991.
14. Cudney, B., Patel, S., Weisgraber, K., Newhouse, Y.,
McPherson, A. Screening and optimization strategies for
macromolecular crystal growth. Acta Crystallogr. D50:
414-423, 1994.
15. Otwinowski, Z. DENZO: An Oscillation Data Processing
Program for Macromolecular Crystallography. New Haven, CT: Yale University, 1993.
16. Collaborative Computational Project, Number 4. The
CCP4 suite: Programs for protein crystallography. Acta
Crvstallom. D50:760-763. 1994.
17. Matthew; B.W. Solvent content ofprotein crystals. J. Mol.
Biol. 33491-497, 1968.
18. Hendrickson, W.A., Horton, J.R., LeMaster, D.M. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): A vehicle for direct
determination of three-dimensional structure. EMBO J .
9:1665-1672, 1990.
19. Glover, I.D., Denny, R.C., Nguti, N.D., McSweeney, S.M.,
Kinder, S.H., Thompson, A.W., Dodson, E.J., Wilkinson,
A.J., Tame, J.R.H. Structure determination of OppA at 2.3
8, resolution using multiple-wavelength anomalous dispersion methods. Acta Crystallogr. D51:39-47, 1995.
Документ
Категория
Без категории
Просмотров
5
Размер файла
293 Кб
Теги
642
1/--страниц
Пожаловаться на содержимое документа