Neurofilament (NF) Assembly; Divergent Characteristics of Human and Rodent NF-L Subunits

• Published in: The Journal of biological chemistry Vol. 273; no. 9; pp. 5101 - 5108
• Main Authors: Carter, J, Gragerov, A, Konvicka, K, Elder, G, Weinstein, H, Lazzarini, R A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 27.02.1998
• Subjects: Amino Acid Sequence; Animals; Aspartic Acid - chemistry; Dimerization; Humans; Intermediate Filaments - metabolism; Models, Molecular; Molecular Sequence Data; Neurofilament Proteins - chemistry; Neurofilament Proteins - genetics; Neurofilament Proteins - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding; Protein Structure, Secondary; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Repetitive Sequences, Nucleic Acid; Sequence Homology, Amino Acid; Species Specificity; Static Electricity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.9.5101

Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation. We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly. However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments. To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast “interaction trap” system. These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation. Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains. We tested these predictions using site-specific mutagenesis. Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble. Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended β-sheet conformation, are consonant with an α-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization. These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species.