Model of the ran-RCC1 interaction using biochemical and docking experiments.
RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.
Azuma
Y
Y
Department of Molecular Biology, Kyushu University, Fukuoka, Japan.
Renault
L
L
García-Ranea
J A
JA
Valencia
A
A
Nishimoto
T
T
Wittinghofer
A
A
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein; D000409:Alanine