Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A(11) molecular alignment mode in keratin intermediate filaments

Both analyses of x-ray diffraction patterns of well oriented specimens of t richocyte keratin intermediate filaments (IF) and in vitro cross-linking ex periments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A(11), A(22), a nd A(12), based on which parts of the major rod domain segments are overlap ped. Here we have examined which residues may be important for stabilizing the A(11) mode. Using the K5/R14 system, we have made point mutations of ch arged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: th e formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg(10) of the 1A rod domain, and the conserved residues Glu(4) and Glu(6) of the l inker L2, were essential for stability. Additionally, conserved residues Ly s(31) of 1A and Asp(1) of 2A and non-conserved residues Asp/Asn(9) of 1A, A sp/Asn(3) of 2A, and Asp(7) of L2 are important for stability. Notably, the se groups of residues lie close to each other when two antiparallel molecul es are aligned in the A(11) mode, and are located toward the ends of the ov erlap region. Although other sets of residues might theoretically also cont ribute, we conclude that these residues in particular engage in favorable i ntermolecular ionic and/or H-bonding interactions and thereby may play a ro le in stabilizing the A(11) mode of alignment in keratin IF.