Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors.
NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes. We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization. Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct. The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 A resolution using synchrotron radiation, whereas the importin-beta-nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 A resolution.
Bayliss
R
R
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
Kent
H M
HM
Corbett
A H
AH
Stewart
M
M
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D028884:Karyopherins; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D029543:Nucleocytoplasmic Transport Proteins; D020449:Repetitive Sequences, Amino Acid; D029701:Saccharomyces cerevisiae Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D028884:Karyopherins; D046911:Macromolecular Substances; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D029701:Saccharomyces cerevisiae Proteins