Abstract
Mutations in intermediate filament protein genes are responsible for a number of inherited genetic diseases including skin blistering diseases, corneal opacities, and neurological degenerations. Mutation of the arginine (Arg) residue of the highly conserved LNDR motif has been shown to be causative in inherited disorders in at least four different intermediate filament (IF) proteins found in skin, cornea, and the central nervous system. Thus this residue appears to be broadly important to IF assembly and/or function. While the genetic basis for these diseases has been clearly defined, the inability to determine crystal structure for IFs has precluded a determination of how these mutations affect assembly/structure/function of IFs. To investigate the impact of mutation at this site in IFs, we have mutated the LNDR to LNDS in vimentin, a Type III intermediate filament protein, and have examined the impact of this change on assembly using electron paramagnetic resonance. Compared with wild type vimentin, the mutant shows normal formation of the coiled coil dimer, with a slight reduction in the stability of the dimer in rod domain 1. Probing the dimer-dimer interactions shows the formation of normal dimer centered on residue 191 but a failure of dimerization at residue 348 in rod domain 2. These data point toward a specific stage of assembly at which a common disease-causing mutation in IF proteins interrupts assembly.
Highlights
The intermediate filament (IF)1 protein gene family consists of about 60 members at present
Mutation of the arginine (Arg) residue of the highly conserved LNDR motif has been shown to be causative in inherited disorders in at least four different intermediate filament (IF) proteins found in skin, cornea, and the central nervous system
In the early 1990s, three lines of evidence independently identified IF protein genes as the site of mutations leading to epidermolysis bullosa simplex (EBS) and other skin blistering diseases in humans such as epidermolysis hyperkeratosis [3, 5, 12,13,14,15,16,17,18,19]
Summary
The intermediate filament (IF) protein gene family consists of about 60 members at present. While the predicted secondary structure of the central rod domain is conserved, there is much primary sequence variability except at two small motifs located at either end of the central rod domain At these sites, sequence conservation has been quite strong. Mehrani et al [27] showed that K14 LNDR mutated to LNDL was unable to form IFs, unable to assemble into existing IF networks in cells, and was significantly altered in the tetramer configuration. Lys LNAR was unable to form filaments in vitro and unable to integrate into IF networks but exhibited slightly less stability than the wild type They summarize the characterization of these substitutions in the Asp and Arg locations of LNDR as significantly destabilizing the two-molecule entities (tetramer)
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