Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate - PubMed (original) (raw)

Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate

P Van Roey et al. EMBO J. 2001.

Abstract

I-TevI is a site-specific, sequence-tolerant intron endonuclease. The crystal structure of the DNA-binding domain of I-TevI complexed with the 20 bp primary binding region of its DNA target reveals an unusually extended structure composed of three subdomains: a Zn finger, an elongated segment containing a minor groove-binding alpha-helix, and a helix-turn-helix. The protein wraps around the DNA, mostly following the minor groove, contacting the phosphate backbone along the full length of the duplex. Surprisingly, while the minor groove-binding helix and the helix-turn- helix subdomain make hydrophobic contacts, the few base-specific hydrogen bonds occur in segments that lack secondary structure and flank the intron insertion site. The multiple base-specific interactions over a long segment of the substrate are consistent with the observed high site specificity in spite of sequence tolerance, while the modular composition of the domain is pertinent to the evolution of homing endonucleases.

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Figures

Fig. 1. I-_Tev_I–DNA interaction. (A) Cartoon of the two-domain structure of I-_Tev_I with its DNA homing site. (B) Protein–DNA contacts. The sequence of the DNA fragment used in the crystal structure determination with interaction sites as identified from ethylation and methylation protection assays indicated as arrows and circles (open, weak; closed, strong), respectively. Bars below indicate the DNA locations of the three types of protein–DNA contacts in the structure, showing the consistency with the biochemical data. P and H correspond to regions involved in phosphate–backbone and hydrophobic contacts, respectively, while B refers to base-specific hydrogen-bonding contacts. IS and CS indicate the insertion and cleavage sites, respectively.

Fig. 2. Electron density map for the Zn finger subdomain. Stereodiagram showing the final (2_F_o – _F_c) map for residues 149–168, contoured at the 1.5σ level. The Zn ion is shown as a blue sphere in the lower center of the view and the 10 residue loop oriented towards the top of the figure. The figure was prepared using SETOR (Evans, 1993).

Fig. 3. Three-dimensional structure of the complex of the DNA-binding domain of I-_Tev_I with its substrate. The complex is shown (A) perpendicular to the DNA axis and (B) along the DNA axis. Distortions to the DNA are limited to widening of the minor groove. (C) Space-filling model of the structure of the complex, showing the continuous tight association between the two molecules. Protein and DNA carbon atoms are colored green and gray, respectively. Figures 3 and 4 were prepared with Molscript (Kraulis, 1991) and Raster3D (Merritt and Bacon, 1997).

Fig. 4. Individual I-_Tev_I–DNA contact regions. (A) Elongated segment between the Zn finger and the minor groove-binding helix. The protein lacks secondary structure but residues 170–180 form a twisted structure that widens the minor groove. Base-specific hydrogen-bonding contacts (red dotted lines) throughout this segment are interrupted by the hydrophobic insertion of the phenyl ring of Phe177 (yellow). (B) The minor groove-binding α-helix. Only one hydrogen-bonding contact is seen in the helix (Ser191 to the phosphate backbone), and the surface close to the DNA consists of three hydrophobic residues. The section between the helix and the helix–turn–helix subdomain includes the remaining base-specific hydrogen-bonding contacts, Asn201 to GUA40 and CYT14. (C) The helix–turn–helix subdomain inserts its second helix into the major groove. Several of the residues of this helix make hydrogen-bonding contacts to the phosphate backbone (red dotted lines), but the surface of the helix adjacent to the DNA is mostly hydrophobic and matches the hydrophobic surface of the DNA, which presents the C5-methyl groups of thymidines 16, 17, 18, 33 and 34. The closest hydrophobic contacts are shown as blue dotted lines.

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Intertwined structure of the DNA-binding domain of intron endonuclease I-TevI with its substrate - PubMed (original) (raw)