Proteolytic cleavage of Gram-positive ß recombinase is required for crystallization

Peter Orth, Petra Jekow, Juan C. Alonso1 and Winfried Hinrichs2

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


    Abstract
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 Abstract
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 Material and methods
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ß Recombinase, a DNA resolvase-invertase, catalyzes in the presence of a chromatin-associated protein such as Hbsu, DNA resolution or DNA inversion on supercoiled substrates containing two directly or inversely oriented target (six) sites. Single crystals of the ß recombinase from plasmid pSM19035 were obtained using the vapor diffusion technique with ammonium phosphate as the precipitating agent. The crystals diffracted X-rays to a maximum resolution of 2.5Å. Due to proteolytic degradation during the crystallization experiment, the crystals contain only the N-terminal catalytic domain of ß recombinase corresponding to about 60% of the molecular mass of the initially assayed native protein. The proteolytic removal of the C-terminal DNA-binding domain demonstrated that protein modification can be essential to provide material suitable for X-ray analysis.

Keywords: catalytic domain/protein crystallization/proteolytic fragment/resolvase-invertase


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
ß Recombinase, a site-specific DNA resolvase-invertase encoded by the Streptococcus pyogenes plasmid pSM19035, belongs to the Tn3/{gamma}{delta} family of recombinases (reviewed by Alonso et al., 1996Go). DNA resolvases and DNA invertases are highly specialized in catalyzing, on a supercoiled DNA substrate, resolution or inversion, respectively (Stark et al., 1992Go; Grindley, 1994Go). The 23.8 kDa ß recombinase (205 amino acid residues) does not have this bias and catalyzes both types of reactions efficiently (Rojo and Alonso, 1994Go; Alonso et al., 1995Go). The ß target site (six site), which is 85 base pairs in length, includes the palindromic crossover site (subsite I) and a non-palindromic essential site (subsite II) (Rojo and Alonso, 1995Go). To each subsite one homodimeric ß recombinase molecule is bound. These binding sites (34 bp each) are separated by a 15 bp linker. For the formation of a synaptic complex the juxtaposition of two such six sites with two bound ß recombinase dimers and an undefined number of chromatin-associated proteins (e.g. Hbsu) is required. These chromatin-associated proteins are supposed to facilitate the formation of the recombination complex; the accessory site(s), to which they bind, are not well defined (Alonso et al., 1996Go). Upon formation of the recombination complex the six sites will be oriented in parallel. Depending on the DNA structure between the two six sites, resolution or inversion takes place. Inversion occurs only on inversely oriented DNA, whereas resolution is independent of the orientation of the six sites. When two six sites for the ß recombinase are directly oriented on a supercoiled DNA molecule, DNA resolution is the only resulting product, whereas at inversely oriented six sites DNA resolution or DNA inversion occurs depending on the degree of supercoiling and on the number of Hbsu molecules bound. Relaxation of the DNA substrate fully inhibits the resolution activity of the ß recombinase at inversely oriented six sites, but does not affect its ability to catalyze inversion reactions. DNA inversion was observed even on linear DNA substrates (Canosa et al., 1998Go).

Different structures of the distantly related site-specific recombinase, {gamma}{delta} DNA resolvase [of an N-terminal chymotryptic fragment (residues 1 to 140) (Sanderson et al., 1990Go), of the DNA-free form (Rice and Steitz, 1994aGo) and of the molecule bound to subsite I (Yang and Steitz, 1995Go)] have been reported. The {gamma}{delta} resolvase homodimer shows a modular structure with a dimerization domain covering 66% of the molecular mass and two long {alpha}-helices with C-terminal three-helix-bundles representing the DNA-binding domains. While in the DNA bound form these long {alpha}-helices, as well as the three-helix-bundles, establish site-specific contacts to the DNA resolvase (Yang and Steitz, 1995Go) or DNA invertase recombination site (Feng et al., 1994Go), they could not be modeled in the DNA-free form of {gamma}{delta} 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 {gamma}{delta} resolvase as the search model.


    Material and methods
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 Abstract
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 Material and methods
 Results and discussion
 References
 
Crystallization

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 Tris–HCl pH 7.5. The protein concentration was determined as previously described (Bradford, 1976Go). The protein solution was screened for crystallization conditions using a matrix crystal screen (Jancarik and Kim, 1991Go) 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, 1996Go) and the programs of the CCP4 suite (CCP4, 1994Go). Crystal and data collection characteristics are summarized in Table IGo.


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Table I. Crystallographic characteristics and summary of data collection
 
The self-rotation function was calculated using POLARRFN (CCP4, 1994Go). Molecular replacement was performed using AMoRe (Navaza, 1994Go) with the resolvase catalytic domain (Rice and Steitz, 1994bGo; PDB entry code 2RSL) as search model. The crystal packing was inspected using the program O (Jones et al., 1991Go). For sequence alignment we used CLUSTAL W (Thompson et al., 1994Go).


    Results and discussion
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Proteolytic degradation

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 SDS–PAGE. According to these SDS–PAGE patterns, the molecular mass of the protein decreased from 23.8 kDa (native protein assayed; 26 kDa in SDS–PAGE) 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 1Go). 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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Fig. 1. MALDI mass-spectrum of dissolved crystals. The molecular masses of the fragments are indicated by the numbers above the peaks. Arrows point to the corresponding cleavage sites in the ß recombinase sequence.

 
The {gamma}{delta} resolvase consists of a N-terminal catalytic domain (residues 1–120), a C-terminal DNA-binding domain with the three-helix-bundle (residues 148–183) and an extended arm region (residues 121–147) that connects the two domains (Yang and Steitz, 1995Go). A comparison of the putative cleavage sites on the recombinase sequence with the respective sites in the crystal structure of {gamma}{delta} resolvase reveals that cleavage might occur in the extended arm region, N-terminal to the long {alpha}-helix and C-terminal to the dimerization domain. Thus, we assume the crystallized fragments contain residues necessary for catalysis, dimerization and cooperativity in binding of the ß recombinase to the 85 bp six site (Canosa et al., 1997Go).

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, 1968Go). The calculated self-rotation function shows that in the {kappa} = 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 2Go). 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 ({Phi} = 22.5°). This orientation of the local twofold axis creates an apparent eightfold symmetry and causes in the {kappa} = 45° section a peak with the same height as observed for the non-crystallographic dyad in the {kappa} = 180° section. The location of this peak coincides with that of the tetragonal axis.



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Fig. 2. Stereographic plot of the {kappa} = 180° section of the self-rotation function for the ß recombinase data (35–4 Å). The arrow indicates one position of the non-crystallographic dyad symmetrically placed between the crystallographic twofold axis.

 
Cross-rotation and translation search for molecular replacement were conducted in the resolution range of 20–4 Å. As search model we defined the homodimeric {gamma}{delta} resolvase catalytic domain [molecules A and B of the PDB entry 2RSL (Rice and Steitz, 1994bGo)] with all side-chains excluding water molecules. The sequence identity between both recombinases is 30% along the dimerization domain. Rotation solutions with the dimer axis coinciding with the non-crystallographic axis were used for calculating the translation function in the two alternative space groups P41212 or P43212, respectively. The best solutions were refined using 10 cycles of rigid body refinement and finally belong to space group P43212.

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 {gamma}{delta} 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 {alpha}-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.


    Acknowledgments
 
We thank María M.Vázquez for the harvesting of cell mass, the scientific staff of LURE (Orsay, Paris) and EMBL (DESY, Hamburg) for assistance with data collection, Peter Franke (FU Berlin) for the MALDI-analysis, Werner Schröder (FU Berlin) for the N-terminal sequencing and Wolfram Saenger for support. This work was partially supported by grants PB 96-0817 from DGCICYT and 06G-004/96 from the Consejería de Educación y Cultura de la Comunidad de Madrid to J.C.A.


    Notes
 
2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
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Received September 4, 1998; revised February 11, 1999; accepted February 11, 1999.





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