Antibodies to Cytoplasmic Dynein Heavy Chain Map the Surface and Inhibit Motility

J Mol Biol. 2001 Apr 13;307(5):1317-27. doi: 10.1006/jmbi.2001.4566.

Abstract

Polyclonal antibodies have been raised against four 16 residue peptides with sequences taken from the C-terminal quarter of the human cytoplasmic dynein heavy chain. The sites are downstream from a known microtubule-binding domain associated with the "stalk" that protrudes from the motor domain. The antisera were assayed using bacterially expressed proteins with amino acid sequences taken from the human cytoplasmic dynein heavy chain. Every antiserum reacted specifically with the appropriate expressed protein and with pig brain cytoplasmic dynein, whether the protein molecules were denatured on Western blots or were in a folded state. But, whereas three of the four antisera recognized freshly purified cytoplasmic dynein, the fourth reacted only with dynein that had been allowed to denature a little. After affinity purification against the expressed domains, whole IgG molecules and Fab fragments were assayed for their effect on dynein activity in in vitro microtubule-sliding assays. Of the three anti-peptides that reacted with fresh dynein, one inhibited motility but the others did not. The way these peptides are exposed on the surface is compatible with a model whereby the dynein motor domain is constructed from a ring of AAA protein modules, with the C-terminal module positioned on the surface that interacts with microtubules. We have tentatively identified an additional AAA module in the dynein heavy chain sequence, which would be consistent with a heptameric ring.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / immunology*
  • Antibodies / pharmacology*
  • Binding Sites
  • Brain
  • Dyneins / antagonists & inhibitors*
  • Dyneins / chemistry
  • Dyneins / immunology*
  • Dyneins / metabolism
  • Humans
  • Immune Sera / immunology
  • Immune Sera / pharmacology
  • Immunoglobulin Fab Fragments / immunology
  • Microscopy, Electron
  • Microscopy, Video
  • Microtubules / drug effects
  • Microtubules / immunology
  • Microtubules / metabolism
  • Microtubules / ultrastructure
  • Molecular Motor Proteins / antagonists & inhibitors
  • Molecular Motor Proteins / chemistry
  • Molecular Motor Proteins / immunology
  • Molecular Motor Proteins / metabolism
  • Molecular Sequence Data
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / immunology
  • Protein Structure, Tertiary
  • Protein Subunits
  • Rabbits
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Swine

Substances

  • Antibodies
  • Immune Sera
  • Immunoglobulin Fab Fragments
  • Molecular Motor Proteins
  • Peptide Fragments
  • Protein Subunits
  • Recombinant Proteins
  • Dyneins