Figure 12.

Analysis of the coiled coil domain in IS2OrfAB aligned with similar domains in the IS3 family. (A) The coiled coil sequence in IS2 identified by the PCOILS analysis of coiled coils [57,58] annotated to show the four putative heptad repeats of a leucine zipper-like motif. Italicized letters a to g represent the repeated positions within each heptad. The critical d positions which favor hydrophobic leucines are highlighted in green (or in red for a non-canonical amino acid). The a-located buried asparagine (N94) is shown in red while green lettering identifies the three canonical a-located hydrophobics. The five randomly induced mutations are indicated by arrows. The corresponding GMF mutant strain is listed beneath each mutation. (B) Alignment of the coiled coil domains of seven members from the five principal subgroups of the IS3 family showing their relationships to the putative heptads of a leucine-zipper motif. Annotation is as described in part A but for the IS2 sequence the a positions are highlighted in aqua. (C) Analysis of the potential of the coiled coil sequence in IS2 to function as a leucine zipper and the effect of mutations recovered within the motif on that function. The data suggest that the sequence which fails the 2ZIP test for a leucine zipper [59] may indeed have that function. Stabilization by the two d-located leucines is indicated by vertical bold green lines, by the a-located hydrophobics by narrow green lines and by the buried asparagine by a vertical broken red line. Weak salt bridges between glutamines in the g and e locations in heptads 1 and 2 are indicated by a large narrow-lined red × and the canonical ionic salt bridges between the g and e-located E and K residues in heptads 3 and 4, are indicated by a large bold red X. Binding efficiencies (see Figure 6) and transposition frequencies (see Table 2) are listed below the schematic. Additional annotation is as described in part A. GFP: green fluorescent protein; IS: insertion sequence.