Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate filaments.
Both analyses of x-ray diffraction patterns of well oriented specimens of trichocyte keratin intermediate filaments (IF) and in vitro cross-linking experiments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A11, A22 and A12, based on which parts of the major rod domain segments are overlapped. Here we have examined which residues may be important for stabilizing the A11 mode. Using the K5/K14 system, we have made point mutations of charged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the 1A rod domain, and the conserved residues Glu4 and Glu6 of the linker L2, were essential for stability. Additionally, conserved residues Lys31 of 1A and Asp1 of 2A and non-conserved residues Asp/Asn9 of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are important for stability. Notably, these groups of residues lie close to each other when two antiparallel molecules are aligned in the A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in favorable intermolecular ionic and/or H-bonding interactions and thereby may play a role in stabilizing the A11 mode of alignment in keratin IF.
Mehrani
T
T
Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752, USA.
Wu
K C
KC
Morasso
M I
MI
Bryan
J T
JT
Marekov
L N
LN
Parry
D A
DA
Steinert
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004173:Dipodomys; D019084:Fluorescent Antibody Technique, Indirect; D007633:Keratins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D014961:X-Ray Diffraction
D017931:DNA Primers; D007633:Keratins