The structure of the Q69L mutant of GDP-Ran shows a major conformational change in the switch II loop that accounts for its failure to bind nuclear transport factor 2 (NTF2).
We report the 2.3 A resolution X-ray crystal structure of the GDP-bound form of the RanQ69L mutant that is used extensively in studies of nucleocytoplasmic transport and cell-cycle progression. When the structure of GDP-RanQ69L from monoclinic crystals with P21 symmetry was compared with the structure of wild-type Ran obtained from monoclinic crystals, the Q69L mutant showed a large conformational change in residues 68-74, which are in the switch II region of the molecule which changes conformation in response to nucleotide state and which forms the major interaction interface with nuclear transport factor 2 (NTF2, sometimes called p10). This conformational change alters the positions of key residues such as Lys71, Phe72 and Arg76 that are crucial for the interaction of GDP-Ran with NTF2 and indeed, solution binding studies were unable to detect any interaction between NTF2 and GDP-RanQ69L under conditions where GDP-Ran bound effectively. This interaction between NTF2 and GDP-Ran is required for efficient nuclear protein import and may function between the docking and translocation steps of the pathway.
Stewart
M
M
MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, CB2 2QH, England.ms@mrc-lmb.cam.ac.uk
Kent
H M
HM
McCoy
A J
AJ
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism*; D028884:Karyopherins; D008958:Models, Molecular; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014981:Xenopus; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D002457:Cell Extracts; D028884:Karyopherins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C091775:ran-binding protein 1; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein