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-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. 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 in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular 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 functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all 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 detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-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.
Wu
K C
KC
Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Bryan
J T
JT
Morasso
M I
MI
Jang
S I
SI
Lee
J H
JH
Yang
J M
JM
Marekov
L N
LN
Parry
D A
DA
Steinert
P M
PM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D006801:Humans; D007382:Intermediate Filaments / Q000648:ultrastructure*; D007633:Keratins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D007633:Keratins