W0098: The Dna-Binding Domain of the Intron-Encoded Endonuclease I-Tevi Harbors Three Different Dna-Binding Motifs

I-TevI is a 28-kDa homing endonuclease encoded by the td intron of bacteriophage T4. The enzyme binds a long (37 base pairs) DNA target, exhibiting high sequence tolerance. I-TevI consists of two functionally distinct domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, connected by a long flexible linker. The isolated DNA-binding domain, residues 130 to 245, contacts a 20 base pair region of the homing site and binds with the same affinity as the full-length protein. We have determined the 2.2 Å crystal structure of the complex of the DNA-binding domain with a DNA duplex that includes its 20 base pair contact region and complementary single base overhangs at the 5'-end of each strand. 95 protein residues, starting at residue 149, are observable in the electron density map. The protein wraps around the DNA, making contacts mainly along the minor groove. It includes three different DNA-recognition motifs connected by long linkers that lack secondary structure. The three motifs are a zinc-finger, an α-helix that binds in the minor groove and a helix-turn-helix motif that contacts the major groove. The zinc-finger, residues 150 to 166, was unexpected since it was not predicted from sequence analysis. The zinc ion is coordinated by four cysteine residues in a tetrahedral arrangement but with an unusual -CXC(X)₁₀CXXC- motif. The minor-groove binding helix, residues 183 to 194, distorts the DNA by widening the groove by about 4 Å. The helix-turn-helix motif is part of the only moiety with a defined three-dimensional fold. This fold comprises the last 40 residues and contains a small 3-stranded antiparallel β-sheet in addition to the helix-turn-helix motif. The protein and DNA make close contacts along their entire lengths. The structural details are consistent with the observed sequence tolerance of the enzyme. Most protein-DNA contacts are to the phosphate backbone but, interestingly, the few base-specific contacts that do occur are in the linkers between the individual motifs.