Analysis of the LAGLIDADG interface of the monomeric homing endonuclease I-DmoI.
The general structural fold of the LAGLIDADG endonuclease family consists of two similar alpha/beta domains (alphabetabetaalphabetabetaalpha) that assemble either as homodimers or monomers with the domains related by pseudo-two-fold symmetry. At the center of this symmetry is the closely packed LAGLIDADG two-helix bundle that forms the main inter- or intra-molecular contact region between the domains of single- or double-motif proteins, respectively. In this work, we further examine the role of the LAGLIDADG residues involved in the helix-helix interaction. The interchangeability of the LAGLIDADG helix interaction was explored by grafting interfacial residues from the homodimeric I-CreI into the corresponding positions in the monomeric I-DmoI. The resulting LAGLIDADG exchange mutant is partially active, preferring to nick dsDNA rather than making the customary double-strand break. A series of partial revertants within the mutated LAGLIDADG region are shown to restore cleavage activity to varying degrees resulting in one I-DmoI mutant that is more active than wild-type I-DmoI. The phenotype of some of these mutants was reconciled on the basis of similarity to the GxxxG helix interaction found in transmembrane proteins. Additionally, a split variant of I-DmoI was created, demonstrating that the LAGLIDADG helices of I-DmoI are capable of forming and maintaining the protein-protein interface in trans to create an active heterodimer.
Silva
George H
GH
Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA.
Belfort
Marlene
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013997:Time Factors
D004247:DNA; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific