Abstract
PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.
Highlights
Nation thereby integrating the homing endonuclease gene and completing the homing process
A comparison of the structures of the apoenzyme and the complex shows that three major conformational changes are evident for the induced fit of enzyme and substrate (Fig. 1): (i) the loop comprising residues 53–70 of domain I, which is located at the interface between the two domains, moves toward the DNA upon complex formation; (ii) the -hairpin loop comprising residues 369 –375 of domain II undergoes a hinge-flap motion relative to the ligand-free structure, and (iii) the 269 –284 loop located at the extreme of the endonuclease domain becomes ordered in the ligand-bound structure
Our results suggest a straightforward mechanism for communication between domains I and II, which is mediated by divalent metal ion binding to the active site in domain II and loop53–70 in domain I closing onto the DNA
Summary
I, intron-encoded homing endonuclease; PI, protein intein homing endonuclease; FRET, fluorescence resonance energy transfer; TMR, tetramethylrhodamine; LC, lower complex; UC, upper complex; Ctr, Candida tropicalis. Homing endonucleases are similar to restriction endonucleases in that they require Mg2ϩ ions for cleavage to generate DNA fragments with a 5Ј-phosphate and a 3Ј-hydroxyl, they are structurally unrelated They can be divided into four families, characterized by common sequence motifs: the LAGLIDADG, His-Cys-box, HN-H, and GIY-YIG. A comparison of the structures of the apoenzyme and the complex shows that three major conformational changes are evident for the induced fit of enzyme and substrate (Fig. 1): (i) the loop comprising residues 53–70 of domain I, which is located at the interface between the two domains, moves toward the DNA upon complex formation; (ii) the -hairpin loop comprising residues 369 –375 of domain II undergoes a hinge-flap motion relative to the ligand-free structure, and (iii) the 269 –284 loop located at the extreme of the endonuclease domain (domain II) becomes ordered in the ligand-bound structure Cleavage of this loop by trypsin at position 277 inactivates the enzyme [24]. Our results suggest a straightforward mechanism for communication between domains I and II, which is mediated by divalent metal ion binding to the active site in domain II and loop in domain I closing onto the DNA
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