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

Biochemistry. 2001 Aug 28;40(34):10342-9. doi: 10.1021/bi0108305.


A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-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 has been shown to be required for filament formation. One or more cross-linked 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 to 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 which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two 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, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • DNA / metabolism*
  • Desmin / chemistry
  • Desmin / metabolism
  • Glial Fibrillary Acidic Protein / chemistry
  • Glial Fibrillary Acidic Protein / metabolism
  • Humans
  • Intermediate Filament Proteins / chemistry*
  • Intermediate Filament Proteins / metabolism*
  • Keratins / chemistry
  • Keratins / metabolism
  • Membrane Glycoproteins*
  • Mice
  • Molecular Sequence Data
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / metabolism
  • Neurofilament Proteins / chemistry
  • Neurofilament Proteins / metabolism
  • Oligodeoxyribonucleotides / chemistry*
  • Oligodeoxyribonucleotides / metabolism*
  • Peripherins
  • Protein Structure, Secondary
  • Protein Subunits
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Swine
  • Vimentin / chemistry
  • Vimentin / metabolism


  • Desmin
  • Glial Fibrillary Acidic Protein
  • Intermediate Filament Proteins
  • Membrane Glycoproteins
  • Nerve Tissue Proteins
  • Neurofilament Proteins
  • Oligodeoxyribonucleotides
  • PRPH protein, human
  • Peripherins
  • Protein Subunits
  • Vimentin
  • neurofilament protein L
  • Keratins
  • DNA