Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-r esidue trigger motif that seems to be required for the stability of the coi led coil. However, as currently defined, the motif is absent from intermedi ate filament (IF) protein chains, which nevertheless form segmented two-cha in coiled coils. Ln the present work, we have searched for and identified t wo regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger mot ifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled i n vitro and in vivo, and the stabilities of two-chain (one-molecule) and tw o-molecule assemblies were examined with use of a urea disassembly assay. O ur new data document that there is a region located between residues 100 an d 113 of the 2B rod domain segment that is absolutely required for molecula r stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu ) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functio ns in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residue s 79 and 91 of the 1B rod domain segment, which may also be conserved in al l IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little dete ctable effect on IF assembly, except for the conserved Lys-23 residue of th e 2B rod domain segment. Cross-linking and modeling studies revealed that L ys-23 may lie very close to Glu-106 when two molecules are aligned in the A (22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.