Crystal structure of intein homing endonuclease II encoded in DNA polymerase gene from hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (2006)

Intein homing endonucleases are bifunctional proteins catalyzing both protein splicing and site-specific DNA double-strand cleavage.1-3 Protein splicing is a posttranslational process involving precise excision of an intervening protein domain, termed an intein. An intein often exhibits site-specific endonuclease activity, which recognizes and cleaves the DNA sequence lacking its coding DNA sequence. The recognition sequences are usually asymmetrical and 12- to 40-bp long.3 Herein, we report the crystal structure of PI-TkoII, an intein endonuclease II from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1. PI-TkoII is a product of the polA gene: mature KOD DNA polymerase.4 To date, crystal structures of intein homing endonucleases have only been described for PI-SceI from Saccharomyces cerevisiae,5, 6 GyrA intein in bacterial gyrase A subunit from Mycobacterium xenopi,7 and PI-PfuI from Pyroccocus furiosus.8 PI-TkoII shares a low sequence similarity to the other inteins including PI-SceI (21.7% identity in 411 amino acids overlap), GyrA intein (34.4% identity in 61 amino acids overlap), and PI-PfuI (22.7% in 322 amino acids overlap). Although the only structure of archaeal intein has been reported for PI-PfuI, molecular masses are very different between PI-TkoII and PI-PfuI, with 62 and 53 kDa, respectively. The minimal recognition sequence for PI-TkoII involves a 16-bp fragment (5′-CAGCTACTACGGTTAC-3′),9 which is relatively short compared with other intein homing endonucleases. Structural information on PI-TkoII provides new insights for mechanisms involved in specific endonuclease activity. In the present study, we also discuss similarities and differences in domain architecture between PI-TkoII, PI-PfuI, and PI-SceI. Crystal structures of native and SeMet PI-TkoII were solved at 2.7- and 2.5-Å resolution, respectively (Table I). Because no obvious structural differences between the native and SeMet PI-TkoII were observed, we describe here crystal structure of SeMet PI-TkoII at 2.5-Å resolution. The final model includes 537 residues, 279 water molecules, six glycerols, and eight sulfates. According to the Structural Classification of Proteins database (SCOP),10 PI-TkoII is a member of the “Hedgehog/intein (Hint) domain” fold. The molecule has an overall size of approximately 100 × 65 × 40 Å, and consists of four distinct domains [Fig. 1(A) ]: the endonuclease domain (Endo: residues 273–432, green), the Hint domain (Hint: residues 1–127 and 496–537, dark blue), domain III (III: residues 433–495, light blue), and domain IV (IV: residues 128–272, pink). Structural comparisons showed that structures of endonuclease and Hint domains were mainly conserved among PI-TkoII, PI-PfuI, and PI-SceI, whereas domains III and IV were not [Fig. 1(A–D)]. Domain III is located at the corresponding position of Stirrup domain in PI-PfuI, and at DRR (DNA recognition region) in PI-SceI. However, it shares no structural similarities with the Stirrup domain of PI-PfuI and DRR domain of PI-SceI [Fig. 1(A–C)]. Moreover, PI-TkoII contains an additional domain depicted as domain IV in Figure 1(A), which was missing in the structures of PI-PfuI and PI-SceI [Fig. 1(B and C)]. Structures of PI-TkoII, PI-PfuI, and PI-SceI. A: Stereo ribbon diagram of PI-TkoII. PI-TkkoII contains the endonuclease, Hint, III, and IV domains colored in green, blue, light blue, and pink, respectively. Active site residues are shown. B: Ribbon diagram of PI-PfuI. C: Ribbon diagram of PI-SceI. D: Domain organization and multiple sequence alignment of PI-TkoII, PI-PfuI, and PI-SceI. The aligned sequences were displayed in ESPript.24 Secondary structures were calculated by DSSP.25 Secondary structure annotations and numberings on top correspond to PI-TkoII. α-Helices are represented by spirals and β-strand by arrows. White lettering boxed with a red background indicates residues that are conserved in all three sequences, and red lettering indicates similar residues. The conserved sequence blocks of inteins are indicated. Despite its low sequence similarity, the structure of the endonuclease domain of PI-TkoII is similar to those of the other homing endonucleases. The size of the domain is approximately 60 × 30 × 25 Å. A search for protein folds using the coordinates of endonuclease domain and protein coordinates in the Protein Data Bank (PDB) with the program DALI11 revealed that 96 proteins were structurally most similar (with a Z-score of 2.0 or higher) to the endonuclease domain of PI-TkoII. The three proteins with the highest structural similarity were: the intron-encoded I-DmoI from Desulfurococcus mobilis12 [2.1 Å of root-mean-square deviation (RMSD) over 150 residues with 25% sequence identity; PDB code: 1B24], PI-SceI5 (3.3 Å of RMSD over 153 residues with 20% sequence identity; PDB code: 1VDE), and PI-PfuI8 (2.4 Å of RMSD over 103 residues with 17% sequence identity; PDB code: 1DQ3). The PI-TkoII endonuclease domain contains two subdomains, which are related to each other by a pseudo two-fold axis. Each subdomain contains an antiparalleled β-sheet motif named LAGLIDADG, depicted as Block C and Block E in Figure 1(D). The catalytically essential residues (Glu288 and Asp383, respectively) are conserved in these motifs [Fig. 1(A)]. The three β-sheets comprising residues 289–308, 330–338, and 417–429 protrude from the molecular surface to form a clustered negative charge surface made by Arg292, Arg295, Lys298, Lys306, Arg330, Arg333, Lys418, and Arg427. These protruded β-sheets are also found in PI-SceI and PI-PfuI, but they vary considerably in length, curvature, and amino acid sequences. These β-sheets, with nonsymmetrical structure may promote insertion into DNA grooves to recognize nonsymmetrical DNA duplexes. The Hint domain is mainly composed of antiparalleled β-sheets, and is approximately 45 × 40 × 25 Å. According to DALI server,11 five proteins were found to have a similar structure (with a Z-score of 2.0 or higher) in their Hint domain. Proteins with the highest structural similarity were PI-PfuI8 (1.8 Å of RMSD over 164 residues with 23% sequence identity; PDB code: 1DQ3), DnaB intein from Synechocystis sp.13 (1.7 Å of RMSD over 135 residues with 27% sequence identity; PDB code: 1MI8), GyrA intein7 (2.7 Å of RMSD over 168 residues with 23% sequence identity; PDB code: 1AM2), the 17-kDa fragment of the Hedgehog domain14 (2.7 Å of RMSD over 168 residues with 23% sequence identity; PDB code: 1AM2), and PI-SceI5 (2.4 Å of RMSD over 129 residues with 13% sequence identity; PDB code: 1VDE). The Hint domain has a typical two subdomain structure, which exhibits a symmetrical architecture. The active site for protein splicing is located at the center of the horseshoe-shaped Hint domain, which contains highly invariant residues such as Ser1, His96, His536, and Asn537 [Fig. 1(A)]. These observations suggest that mechanisms of protein splicing are highly conserved among this family. In contrast, domain III has no structural similarity to the other known protein structures. PI-PfuI and PI-SceI contain DNA binding domains, named Stirrup and DRR domains, respectively; and domain III is located at positions corresponding to Stirrup and DRR domains [Fig. 1(A–C)]. Domain III is likely to be involved in DNA recognition. The exposed concaved surface found in domain III is highly positively charged, as depicted by a circle in Figure 2(A). Side-chains of the charged residues such as Glu454, Lys453, and Lys466 protrude from the concave surface, which faces the same side of the adjacent endonuclease active site. A DNA binding model based on the structure of PI-SceI complexed with a DNA duplex6 shows that a target DNA will fit well into the surface, with the minor groove of the DNA duplex [Fig. 2(C)]. Distance between the side-chain of Arg292 on the protruded loop of the endonuclease domain and the side-chain of Lys453 on the surface of domain III is 51 Å, which approximately corresponds to the 16-bp double-stranded DNA. A: Electrostatic charge distribution on the surface of PI-TkoII; the view is projected in the same orientation as Figure 1(A). B: View is rotated by 180° with respect to a vertical axis. C: Docking model for the protein–DNA complex. The minimal 16-bp DNA recognition sequence is shown as a ribbon. Domain IV is approximately 40 × 25 × 20 Å, and its surface is also highly positively charged. According to DALI server,11 proteins with the closest structural homologies in domain IV are putative DNA binding proteins such as the predicted transcription regulator (3.9 Å of RMSD over 70 residues with 14% sequence identity; PDB code: 1XMA), Esa1 histone acetyltransferase fragment15 (3.7 Å of RMSD over 64 residues with 3% sequence identity; PDB code: 1FY7), and the cell division control protein 6 (Cdc6)16 (7.3 Å of RMSD over 75 residues with 12% sequence identity; PDB code: 1FNN). Because the corresponding domain of Cdc6 has been reported to be highly conserved, and to possibly be a DNA binding element,16 domain IV of PI-TkoII might have the potential to bind to DNA. Many positive-charged residues such as Lys159, Lys161, Arg186, Arg187, Arg190, Arg199, Lys201, Lys202, Lys233, Lys256, Lys259, Arg267, and Arg269 are located on the surface of domain IV [depicted as a circle in Fig. 2(B)]. However, the surface is facing the other side of the endonuclease active site [depicted as a circle in Fig. 2(B)]. One possible hypothesis is that this positive-charged surface participates in binding to DNA in cases when target DNA length is much longer than that of the minimal recognition sequence. In that case, DNA bending would be induced by protein–DNA interactions similar to those observed in PI-SceI complexed with DNA.6 Modeling analysis showed that DNA bending angle was greater than that in the PI-SceI and DNA complex; therefore, the DNA recognition mechanism of PI-TkoII might be different from that of PI-SceI. PI-TkoII, an intein endonuclease II from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1; PI-SceI, intein endonuclease from S. cerevisiae; PI-PfuI intein endonuclease from Pyroccocus furiosus; MAD, multiwavelength anomalous dispersion; SeMet, selenomethionine. X-ray diffraction data of the native crystal have been obtained previously.17 To produce selenomethionine substituted (Se-Met) PI-TkoII, cells were cultivated in minimal medium containing Se-Met. Purification and crystallization procedures for Se-Met PI-TkoII were essentially the same as those used for the native protein. The microseeding method was used to optimize reproducibility for Se-Met PI-TkoII. Crystals of Se-Met PI-TkoII began to develop after a week, and growth was completed after a month at 293 K. X-ray diffraction data for SeMet crystals were collected with the synchrotron radiation source of SPring-8. SeMet PI-TkoII crystals belong to the orthogonal space group C2221 with lattice constants of a = 103.97, b = 150.54, c = 145.49 Å (Table I). Multiple-wavelength anomalous diffraction (MAD) data were collected using three different wavelengths (0.9792, 0.9794, and 0.9686 Å). Reflections were indexed, integrated, and scaled DENZO and SCALEPACK.18 Phase calculations were performed using CNS.19 Model building and refinement of native and SeMet PI-TkoII structure were done using the programs O20 and CNS.19 Refinement statistics are shown in Table I. The DNA docking model was prepared by manual docking based on the crystal structure of PI-SceI in complex with DNA6 and then energy minimized with the program CNS.19 Figures were generated by MOLSCRIPT,21 RASTER3D,22 GRASP,23 and ESPRIPT.24 Atomic coordinates and structure factors are available from the PDB under accession code 2CW7 for native and 2CW8 for SeMet PI-TkoII, respectively.