Configuration of the catalytic GIY-YIG domain of intron endonuclease I-Tevl: coincidence of computational and molecular findings

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is fol ded into two structural and functional domains, an N-terminal catalytic dom ain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis and NMR spectroscopy to define the configuration of the N-terminal domain a nd its relationship to the flexible linker, The catalytic domain is an alph a/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structur ed domain corresponds precisely to the GIY-YIG module defined by sequence c omparisons of 57 proteins including more than 30 newly reported members of the family, Although much of the unstructured linker is not essential for a ctivity, residues 93-116 are required, raising the possibility that this re gion may adopt an alternate conformation upon DNA binding. Two invariant re sidues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, ha ve properties of catalytic residues. Furthermore, the GIY-YIG sequence elem ents for which the module is named form part of a three-stranded antiparall el beta-sheet that is important for I-TevI structure and function.