Sites of nucleic acid binding in type I-IV intermediate filament subunit proteins

A combination of enyzmatic and chemical ladder sequencing of photo-cross-li nked protein-single-stranded oligodeoxyribonucleotide complexes and analysi s by MALDI-TOF mass spectrometry was employed to identify the amino acid re sidues responsible for the stable binding of nucleic acids in several inter mediate filament (IF) subunit proteins. The IF proteins studied included th e type I and type II cytokeratins K8, K18, and K19; the type III proteins d esmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; an d the type IV neurofilament triplet protein L (NF-L). The site of nucleic a cid binding was localized to the non-a-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that ha s been shown to be required for filament formation. One or more cross-linke d residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears t o be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in whic h a large number of cross-linked residues were found scattered throughout t he first half of the head domain. Control experiments were also done with t wo bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe (183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, we re in good agreement with literature data. Actin and tubulin could not be c ross-linked to the oligonucleotide. Aside from the insight into the biologi cal activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-n ucleic acid interactions.