Epitope mapping and direct visualization of the parallel, in-register arrangement of the double-stranded coiled-coil in the NuMA protein.
NuMA, a 238 kDa protein present in the nucleus during interphase, translocates to the spindle poles in mitosis. NuMA plays an essential role in mitosis, since microinjection of the NuMA SPN-3 monoclonal antibody causes mitotic arrest and micronuclei formation. We have mapped the approximate position of the epitopes of six monoclonal NuMA antibodies using recombinant NuMA fragments. The SPN-3 epitope has been located to residues 255-267 at the C-terminus of the first helical subdomain of the central rod domain and several residues crucial for antibody binding have been identified. To gain insight into the ultrastructure of NuMA, several defined fragments, as well as the full-length recombinant protein, were expressed in Escherichia coli and purified to homogeneity. They were then characterized by chemical cross-linking, circular dichroism spectra and electron microscopy. The results directly reveal the tripartate structure of NuMA. A long central rod domain is flanked by globular end domains. The rod is 207 nm long and is at least 90% alpha-helical. It reflects a double-stranded coiled-coil with the alpha-helices arranged parallel and in register. The NuMA protein thus forms the longest coiled-coil currently known. Our analyses reveal no indication that recombinant NuMA assembles into filaments or other higher order structures.
Harborth
J
J
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Goettingen, Germany.
Weber
K
K
Osborn
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000235:genetics / Q000648:ultrastructure; D018797:Cell Cycle Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015394:Molecular Structure; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000648:ultrastructure; D008941:Spindle Apparatus / Q000276:immunology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D001324:Autoantigens; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins