Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly

Abstract

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.

Figure 1

Ribbon representation of TkRadB. α-Helices and β-strands are denoted by H1–H7 and S1–S9, respectively, and the N- and C-terminals by N and C, respectively. The L1 and L2 loops are also labelled. Side-chains of Lys-33, Thr-34 and Asp-112 are presented as ball-and-stick models to show the location of the ATP binding site; the first two of the Walker A motif are in green and the last one of the Walker B motif in cyan.

Figure 2

Sequence alignment of TkRadB with homologous proteins, Pyrococcus furiosus RadA (PfRadA), human Rad51 (HsRad51), human Dcm1 (HsDcm1) and E.coli RecA (EcRecA). The Walker A and B motifs are indicated by green and cyan boxes, respectively. The secondary structure elements of TkRadB are shown above the alignment with the same notation as in Figure 1. Similarly indicated are the L1 and L2 loop regions.

Figure 3

Structures of the asymmetric unit of the type 1 crystal (a) and the type 2 crystal (b). Side-chains of Lys-33, Thr-34 and Asp-112 in each monomer are presented as ball-and-stick models as in Figure 1. In (b), the non-crystallographic 2-fold rotation axis is perpendicular to the figure plane.

Figure 4

Surface electrostatic potentials of TkRadB in the type 1 crystal. Acidic and basic regions are coloured red and blue, respectively. (a) Two orthogonal views of the monomer (molecule 1 in Figure 3a). Positions of the residues involved in the intermolecular interface are labelled by the one-letter amino acid code and the sequence number (see text); the arrowhead indicates the ATP binding site. (b) Dimer in the asymmetric unit oriented as in Figure 3a.

Figure 5

ATP-binding site of TkRadB with an AMP–PNP molecule adapted by superposition. (a) Superposition of the AMP-PNP-bound MvRadA molecule (PDB ID:1T4G; yellow) to the TkRadB molecule (molecule 1 of the type 1 dimer; blue). Two protein molecules are presented as Cα-tracing tube models. (b) The ATP-binding site of the TkRadB type 1 structure with the AMP–PNP molecule. The TkRadB monomers are in a ribbon presentation; molecule 1 is in blue and molecule 2 in green. Side-chains of amino acid residues within 6 Å from the AMP–PNP molecule are presented as sticks. Several residues of interest are labelled by each one-letter code, sequence number and chain ID (see text). The AMP–PNP molecule is presented as a magenta stick model in both figures.

Figure 6

Stereo-drawings of the helical assembly of TkRadB in ribbon representation. Each TkRadB monomers are in different colours. Yellow and red segments in each monomer indicate the L1 and L2 loops, respectively.

Figure 7

Surface electrostatic potentials of the helical assembly of TkRadB. The helix model in surface representation is viewed perpendicular to the helix axis (a), from the top of the assembly (b) and from the bottom (c). Acidic and basic regions are coloured red and blue, respectively. The locations of the ATP-binding site and the positively charged belt (see text) are indicated by arrows.

Figure 8

Electron micrograph of the TkRadB filament which was formed during the incubation at 60°C for 15 min. White arrowheads indicate periodic changes in the filament diameter. The scale bar represents 100 nm.

Figure 9

Structural comparison of monomers in the type 1 dimer. (a) Comparison of structural differences between monomers in the type 1 and type 2 crystal structures. Distances between the Cα atoms of the corresponding amino acid residues in two monomers are plotted against the residue number for the two structures (blue for type 1 and yellow for type 2). The L1 and L2 loop regions and the residues showing exceptionally large deviations are labelled. (b) Superposition of two monomers in the type 1 dimer. Molecule 1 and 2 are presented in blue and green Cα-traces, respectively. Side-chains of amino acid residues within 4 Å from the adjacent monomer are presented as sticks. The AMP–PNP molecule in a magenta stick model is placed in the ATP-binding site of molecule 1 as in Figure 5. The L1 and L2 loops and several residues of interest are labelled (see text).