Harborth J, Weber K, Osborn M
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Goettingen, Germany.
EMBO J. 1995 Jun 1;14(11):2447-60. doi: 10.1002/j.1460-2075.1995.tb07242.x.
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.
核有丝分裂器蛋白（NuMA）是一种在间期存在于细胞核中的238 kDa蛋白质，在有丝分裂时会转移到纺锤体极。NuMA在有丝分裂中起着至关重要的作用，因为注射NuMA SPN - 3单克隆抗体可导致有丝分裂停滞和微核形成。我们使用重组NuMA片段绘制了六种单克隆NuMA抗体表位的大致位置。SPN - 3表位已定位到中央杆结构域第一个螺旋亚结构域C末端的255 - 267位残基，并且已鉴定出几个对抗体结合至关重要的残基。为了深入了解NuMA的超微结构，在大肠杆菌中表达了几个特定的片段以及全长重组蛋白，并纯化至同质。然后通过化学交联、圆二色光谱和电子显微镜对它们进行表征。结果直接揭示了NuMA的三重结构。一个长的中央杆结构域两侧是球状末端结构域。杆长207 nm，至少90%为α螺旋。它反映了一种双链卷曲螺旋结构，α螺旋平行排列且对齐。因此，NuMA蛋白形成了目前已知最长的卷曲螺旋。我们的分析没有表明重组NuMA会组装成细丝或其他高阶结构。
