Dynamic and equilibrium studies on the interaction of Ran with its effector, RanBP1.
Ran, a small nuclear GTP-binding protein, is one of the most abundant Ras-related proteins in eucaryotic cells. Ran is essential for nucleo-cytoplasmatic transport and is primarily localized in the nucleus and at the nuclear pore complex. Here, we characterize the kinetics and equilibrium of the interaction between Ran and RanBP1 by two independent biophysical approaches: fluorescence spectroscopy using analogues of guanine nucleotides and surface plasmon resonance in the BIAcore system. Both approaches result in kinetic and equilibrium data which are in good agreement with each other. Affinities of RanBP1 for Ran in the GTP-bound state were in the nanomolar range, while Ran.GDP bound RanBP1 with a dissociation constant around 10 microM. Interestingly, the difference in affinity of RanBP1 for Ran.GDP was mostly due to a dramatic increase of the dissociation rate constant. Mutant Ran protein lacking the last five amino acids of the C-terminus (RanDeltaC) is unable to facilitate nuclear import in vitro and does not bind to RanBP1. Here, we show that RanBP1 binds RanDeltaC.mGppNHp with KD values around 10 microM, as is the case for its association with full-length Ran.GDP. The loss of affinity of RanBP1 for the triphosphate form of RanDeltaC was a result of both a decrease of the association rate and a moderately increased dissociation of the RanDeltaC.RanBP1 complex. Circular dichroism spectra indicate significant changes in the secondary structure of either Ran.GppNHp, RanBP1, or both proteins upon forming a stable complex with each other.
Kuhlmann
J
J
Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany.
Macara
I
I
Wittinghofer
A
A
D001692:Biological Transport; D015374:Biosensing Techniques; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020931:ran GTP-Binding Protein
D009687:Nuclear Proteins; C091775:ran-binding protein 1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein