Institut für Kristallographie, Freie Universität Berlin, Takustraße 6, 14195 Berlin, Germany and 1 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, C.S.I.C., Campus de la Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
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Abstract |
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Keywords: catalytic domain/protein crystallization/proteolytic fragment/resolvase-invertase
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Introduction |
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Different structures of the distantly related site-specific recombinase, DNA resolvase [of an N-terminal chymotryptic fragment (residues 1 to 140) (Sanderson et al., 1990
), of the DNA-free form (Rice and Steitz, 1994a
) and of the molecule bound to subsite I (Yang and Steitz, 1995
)] have been reported. The
resolvase homodimer shows a modular structure with a dimerization domain covering 66% of the molecular mass and two long
-helices with C-terminal three-helix-bundles representing the DNA-binding domains. While in the DNA bound form these long
-helices, as well as the three-helix-bundles, establish site-specific contacts to the DNA resolvase (Yang and Steitz, 1995
) or DNA invertase recombination site (Feng et al., 1994
), they could not be modeled in the DNA-free form of
resolvase. Here we report the crystallization of the N-terminal catalysis and subunit assembly domain of ß recombinase and the solution of its crystal structure by molecular replacement using the N-terminal catalytic and dimerization domain of
resolvase as the search model.
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Material and methods |
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The ß recombinase was overproduced and purified according to Rojo et al. (1993) and concentrated to 10 mg/ml using Centrex UF2 Microconcentrators with a cut-off of 10 kDa (Schleicher and Schuell) in 600 mM NaCl, 50 mM TrisHCl pH 7.5. The protein concentration was determined as previously described (Bradford, 1976). The protein solution was screened for crystallization conditions using a matrix crystal screen (Jancarik and Kim, 1991
) in the vapor diffusion technique (`hanging drop' mode). The ß recombinase solution and the reservoir solution were mixed at a ratio of (1:1) and allowed to equilibrate at room temperature over 700 µl of reservoir solution. Best crystals were finally obtained from protein solutions with 1 mg/ml of protein and a precipitating solution of 2 M (NH4)2HPO4. Bipyramidal shaped crystals appeared after 6 months with dimensions of 0.5 mmx0.2 mmx0.2 mm. Trials to reduce the crystallization time by increasing the concentrations of precipitant and/or protein resulted in precipitation of amorphous protein material.
X-Ray data collection and analysis
X-Ray diffraction data were collected at 4°C using 345 mm diameter MAR Research imaging plate detectors on synchrotron beam lines DW-32 (LURE, Paris) and BW7B (EMBL outstation at DESY, Hamburg). The images were evaluated using DENZO (Otwinowski and Minor, 1996) and the programs of the CCP4 suite (CCP4, 1994
). Crystal and data collection characteristics are summarized in Table I
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Results and discussion |
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The long crystallization time and our inability to shorten this time by varying precipitant or protein concentration led us to speculate that, cleavage of the protein while assayed could be the precondition for crystallization. Therefore, crystals were dissolved and investigated by SDSPAGE. According to these SDSPAGE patterns, the molecular mass of the protein decreased from 23.8 kDa (native protein assayed; 26 kDa in SDSPAGE) to 14.5 kDa (crystallized protein). The N-terminal protein sequence analysis of the 14.5 kDa polypeptide revealed the same N-terminal sequence as that of the 23.8 kDa non-truncated ß recombinase. The molecular mass of the cleaved protein, further determined by matrix-assisted laser desorption ionisation (MALDI) mass spectroscopy, revealed that the samples contain a mixture of polypeptides with molecular masses between 14.181 and 15.403 Da. The cleavage sites coming into question to produce such fragments lie between residues Ile125 and Lys137 (Figure 1). At present we cannot decide whether proteolysis during or prior the crystallization experiments was due to the presence of a contaminating protease or to autocleavage of the protein.
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Preliminary determination of the crystal structure
Assuming the molecular mass of one ß recombinase catalytic domain dimer per asymmetric unit, the solvent content in the crystal corresponds to 46% (Matthews, 1968). The calculated self-rotation function shows that in the
= 180° section the highest peaks belong to the 4/mmm symmetry due to a set of crystallographic twofold axes perpendicular to the tetragonal axis (Figure 2
). Also perpendicular to the fourfold axis, peaks appear with about 80% of the main intensity corresponding to a non-crystallographic dyad located symmetrically between the other twofold axes (
= 22.5°). This orientation of the local twofold axis creates an apparent eightfold symmetry and causes in the
= 45° section a peak with the same height as observed for the non-crystallographic dyad in the
= 180° section. The location of this peak coincides with that of the tetragonal axis.
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Crystal packing
The catalytic domain of the ß recombinase crystallizes in space group P43212 with the tetragonal axis parallel to the long axis of the unit cell. Perpendicular to this fourfold axis are two crystallographic axes and according to the self-rotation function one additional non-crystallographic axis appears due to the local dimer axis of the catalytic domain.
In the non-truncated resolvase the C-terminal end forms the DNA-binding domain with a long helix and an adjacent three-helix bundle. Sequence-alignment and secondary structure prediction for the putative DNA-binding domain of the ß recombinase reveal most probably the same fold.
The ß recombinase catalytic domain dimers form several crystal packing contacts except the C-terminal parts of the molecules. The different lengths of the polypeptides due to different sites of proteolytic cleavage have thus no influence on the crystal packing. These C-terminal ends form -helices which are solvent-exposed. The observed crystal form is incompatible with the complete ß recombinase molecule as the native C-terminal end would lead to collision in the crystal packing. Therefore, only the processed 14.5 kDa ß recombinase is able to crystallize under the described conditions, the cleavage is a precondition for successful crystallization.
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Acknowledgments |
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Notes |
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References |
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Received September 4, 1998; revised February 11, 1999; accepted February 11, 1999.