Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI.
Small GTPases of the Rho family regulate cytoskeleton remodeling, cell polarity, and transcription, as well as the cell cycle, in eukaryotic cells. Membrane delivery and recycling of the Rho GTPases is mediated by Rho GDP dissociation inhibitor (RhoGDI), which forms a stable complex with prenylated Rho GTPases. We analyzed the interaction of RhoGDI with the active and inactive forms of prenylated and unprenylated RhoA. We demonstrate that RhoGDI binds the prenylated form of RhoA·GDP with unexpectedly high affinity (K(d) = 5 pm). The very long half-life of the complex is reduced 25-fold on RhoA activation, with a concomitant reduction in affinity (K(d) = 3 nm). The 2.8-Å structure of the RhoA·guanosine 5'-[β,γ-imido] triphosphate (GMPPNP)·RhoGDI complex demonstrated that complex formation forces the activated RhoA into a GDP-bound conformation in the absence of nucleotide hydrolysis. We demonstrate that membrane extraction of Rho GTPase by RhoGDI is a thermodynamically favored passive process that operates through a series of progressively tighter intermediates, much like the one that is mediated by RabGDI.
Department of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St. Lucia, Queensland 4072, Australia.
Zakir
Z
Tnimov
Zhong
Z
Guo
Yann
Y
Gambin
Uyen T T
UT
Nguyen
Yao-Wen
YW
Wu
Daniel
D
Abankwa
Anouk
A
Stigter
Brett M
BM
Collins
Herbert
H
Waldmann
Roger S
RS
Goody
Kirill
K
Alexandrov
D000595:Amino Acid Sequence; D001483:Base Sequence; D003460:Crystallization; D017931:DNA Primers; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017368:Protein Prenylation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
C569638:ARHGDIA protein, human; D017931:DNA Primers; D020730:Guanine Nucleotide Dissociation Inhibitors; D062948:rho Guanine Nucleotide Dissociation Inhibitor alpha; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein