Structure of a hexameric form of RadA recombinase from Methanococcus voltae

Hexameric rings of RadA recombinase from M. voltae have been crystallized. Structural comparisons suggest that homologues of RadA tend to form double-ringed assemblies.


Cloning, protein preparation and crystallization
The open reading frame of residues 61-322 of RadA from M. voltae was inserted between the NdeI and XhoI sites of pET28a (Novagen). The resulting plasmid was verified by DNA sequencing using T7 promoter and terminator primers. The recombinant Á 60 MvRadA was overexpressed in E. coli BL21 Rosetta2 (DE3) cells (Novagen) at 310 K for 4 h using 0.5 mM isopropyl -d-1-thiogalactopyranoside as the inducer. The cells were disrupted by sonication. The insoluble particles were removed by centrifugation at 12 000g. Soluble proteins were first separated by nickel-affinity chromatography. The polyhistidine tag was then removed by overnight digestion with 1:100(w:w) thrombin (Sigma-Aldrich) at 294 K. Gel-filtration chromatography was performed with a Sephacryl S-300 HR column (GE Healthcare) using a buffer composed of 0.5 M sodium acetate and 30 mM Tris-HCl pH 7.9. The purified protein was concentrated to $30 mg ml À1 by ultrafiltration.

Crystallization of D 60 MvRadA and diffraction data collection
Á 60 MvRadA crystals (space group C2) were grown by the hangingdrop method and grew to maximum dimensions of 0.4 Â 0.3 Â 0.2 mm. The optimal well solution consisted of 33% polyethylene glycol 400, 1.0 M NaNO 3 , 50 mM MES-NaOH buffer pH 6.7 and 0.06% thymol. A crystal was transferred into the well solution, looped out of the solution and frozen in a nitrogen cryostream at 100 K. The diffraction data set was collected and processed using a Bruker PROTEUM R system at the Saskatchewan Structural Sciences Centre (at a wavelength of 1.5418 Å ). The statistics of the diffraction data are listed in Table 1.

Structural determination and refinement
The previously solved RadA model (PDB entry 1t4g; Wu et al., 2004) was used as the search model for molecular replacement using Phaser (McCoy et al., 2007). Six monomers were located in the asymmetric unit, which is consistent with the existence of noncrys-tallographic sixfold rotational symmetry. The model was iteratively rebuilt using XtalView (McRee, 1999) and refined using CNS (Brü nger et al., 1998) and REFMAC (Murshudov et al., 2011). Statistics of the refinement and model geometry are given in Table 1. 90.6% of nonglycine residues fell in the most favoured region of the Ramachandran plot. No residues were found in the disallowed region. The electron-density map was generated by Coot  and rendered by Raster3D (Bacon & Anderson, 1988). The ribbon and electrostatic surface figures were rendered using CCP4MG (Potterton et al., 2004). C traces were generated by MolScript (Kraulis, 1991) and Raster3D. The coordinates and structure factors have been deposited in the Protein Data Bank (Bernstein et al., 1977;Berman et al., 2000Berman et al., , 2003 with code 4dc9.

Results
3.1. The overall structure of a hexameric form of D 60 MvRadA As in RecA orthologues, a polymerization motif centred at Phe74 contributes to the oligomerization of MvRadA (Wu et al., 2004). In

Figure 1
Electron-density map of the P-loop. The final -weighted 2F o À F c map contoured at 1.2 is shown in stereo. Two putative nitrate-binding sites are also shown. C, N and O atoms are shown in yellow, blue and red, respectively.

Figure 3
The conformational changes of MvRadA. Three C traces are shown in stereo. The Á 60 MvRadA structure is shown in green, except for its C trace from 61 to 75 and from 256 to 285 (magenta). The previously determined ATPase-active filament of MvRadA is shown in cyan. The Á 62 MvRadA structure in the inactive filament is shown in yellow.
order to test the effect of this motif on the oligomerization of MvRadA, we made a series of truncation mutants of MvRadA that lacked 60-65 N-terminal residues. Similar to the previously studied Á 62 MvRadA (Galkin et al., 2006), Á 60 MvRadA is active in hydrolyzing ATP in the presence of poly(dT) 36 but inactive in promoting DNA strand exchange (data not shown). The crystal structure of Á 60 MvRadA was solved by the molecular-replacement method. The ATP-binding P-loop (residues Gly105-Thr112) was ordered with two putative nitrate ions (Fig. 1), consistent with the requirement for a high concentration of sodium nitrate in the crystallization solution.
The peptide chain was largely ordered except for residues 261-268 in the DNA-interacting L2 region (residues Asn256-Arg285). The six monomers of Á 60 MvRadA formed a closed ring with approximate sixfold rotational symmetry (Fig. 2a). The central channel is lined by L1 regions (residues Arg218-Arg230) and has a diameter of 10 Å .

Two-ringed assembly
Crystal-packing analysis as well as the self-rotation function indicated that the Á 60 MvRadA hexamers further packed into face-toface two-ring assemblies with D6 point-group symmetry (Fig. 2a), with one of the six twofold axes coinciding with the crystallographic twofold axis. During the gel-filtration stage of the purification of the RadA proteins, the full-length MvRadA as well as the truncated protein eluted predominantly as a species with a molecular weight of around 200 kDa. As such, the Á 60 MvRadA protein is likely to exist as single rings in solution.

Conformational change of MvRadA
The a polymerization motif centred around a hydrophobic residue (Phe64 in MvRadA) which protrudes into a hydrophobic pocket in an adjacent monomer in the recombinase polymer. In comparison with the previously determined ATPase-active filament (104-105 Å pitch) of MvRadA (PDB entry 2fpm; Wu et al., 2005;Qian et al., 2005) and the Á 62 MvRadA filament (PDB entry 2gdj) with a shorter pitch (91 Å ; Galkin et al., 2006), residues 61-75 showed the most noticeable translation (Fig. 3). This region also contains a conserved Arg74 residue which has been shown to be important for the conformational flexibility of RadA and Rad51 (Chen et al., 2007). In all previously determined filamentous structures of RadA from M. voltae (Wu et al., 2004Qian et al., 2005Qian et al., , 2006Qian et al., , 2007Galkin et al., 2006;Li et al., 2009b) and M. maripaludis (MmRadA; Li et al., 2009a), this Arg74 residue forms a salt bridge with Glu96 (yellow and cyan structures in Fig. 3). In the hexameric Á 60 MvRadA structure the side chain of Arg74 was observed in a noticeably different conformation (green side chain in Fig. 3) that is incapable of retaining the salt bridge. Two recurrent conformations have been observed in the previously determined MvRadA and MmRadA helical structures. One (cyan structure in Fig. 3) is largely ordered except for residues 261-268 in the L2 region and is likely to correspond to the ATPase-active conformation . The other (yellow structure in Fig. 3) is more disordered in the L2 region and is likely to correspond to the 'inactive' post-ATP hydrolysis conformation . The conformation of each RadA monomer in the hexameric form (green and magenta structures in Fig. 3) clearly resembles the ATPase-active form (cyan structure in Fig. 3). A short helix (residues Gly275-Ala282) was observed in the L2 region which corresponds to helix G in EcRecA (Story et al., 1992;De Zutter et al., 2001).

Similar assemblies of PfRadA and HsDMC1
Interestingly, the crystal packing of heptameric PfRadA (Shin et al., 2003; PDB entry 1pzn) and octameric HsDMC1 (Kinebuchi et al., 2004; PDB entry 1v5w) suggests that they both form similar tworinged assemblies with D7 and D8 point-group symmetry (Figs. 2b and 2c), respectively. These assemblies resemble the face-to-face double rings observed for HsDMC1 and SsRadA in the presence of dsDNA by electron microscopy (Passy et al., 1999;Masson et al., 1999;Yang et al., 2001). In all such assemblies the L1 regions (residues Arg218-Arg230 of MvRadA) line a central channel. Each L1 region has three conserved arginine residues (Arg218, Arg224 and Arg230 in MvRadA) in RadA/Rad51/DMC1 proteins. As a result, the central channels of such assemblies are highly positively charged (the central blue regions in Fig. 4).

Discussion
Unlike the structures of filamentous MvRadA, the hexameric Á 60 MvRadA structure revealed a different conformation of Arg74 which is no longer capable of retaining the salt bridge to Glu96. As such, the crystal structure of Á 60 MvRadA further supports the notion that the residue equivalent to Arg74 of MvRadA modulates the conformational changes which give rise to flexibility in the protein assemblies of orthologous proteins (Chen et al., 2007).
The conformational similarity of Á 60 MvRadA and the ATPaseactive form of MvRadA in the helix G region suggests that this short helix is inherently stable. In the structures of filamentous MvRadA and MmRadA disorder of helix G has been correlated with either the presence of ADP or the absence of proper cationic bridging between the C-terminal carbonyl groups of helix G and the -phosphate of the ATP analogue. This structural feature of Á 60 MvRadA is consistent with the notion that ATP hydrolysis in recombinase filaments triggers disorder of helix G and the larger L2 region Li et al., 2009a).
We recently observed that polyanionic compounds such as metatungstate could inhibit MvRadA (Li et al., 2009b) by competing with DNA for positively charged L1 regions lined along an axial groove in the MvRadA filament. Although there is no evidence that such tworinged assemblies exist in solution in the absence of DNA, their highly cationic cavities suggest that anionic compounds that replace DNA could stabilize such recombinase assemblies and thus inhibit the formation of active recombinase filament. It has been discovered that tumour cells tend to have an elevated level of Rad51 expression, which correlates with their resistance to radiotherapy and chemotherapy (Klein, 2008). Therefore, a Rad51 inhibitor could serve as a potential adjuvant for cancer therapy. In addition to suppressing the ATPase activity (Wigle et al., 2006(Wigle et al., , 2009Wigle & Singleton, 2007;Sexton et al., 2010), mimicking the polymerization motif (Cline et al., 2007) and blocking the DNA-binding groove in the recombinase filament (Li et al., 2009b), the two-ringed assemblies of RadA and DMC1 proteins suggest a fourth strategy for inhibiting the recombinase activities of RecA orthologues.