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. 2012 Aug 3;287(32):26549-62.
doi: 10.1074/jbc.M112.371294. Epub 2012 May 24.

Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI

Zakir Tnimov  1 , Zhong Guo, Yann Gambin, Uyen T T Nguyen, Yao-Wen Wu, Daniel Abankwa, Anouk Stigter, Brett M Collins, Herbert Waldmann, Roger S Goody, Kirill Alexandrov
Affiliations

Quantitative analysis of prenylated RhoA interaction with its chaperone, RhoGDI

Zakir Tnimov et al. J Biol Chem. .

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
Interaction analysis of RhoGDI with unprenylated RhoA. A, representative ITC data for titration of 10 μm RhoGDI with either 160 μm RhoA·GDP (filled squares) or buffer (open squares). Solid curve represents a fit of the data to a 1:1 stoichiometry binding model with a calculated Kd value 213 nm; B as in A except that RhoGDI was titrated with 130 μm RhoA·GMPPNP (filled squares) or buffer (open squares). The fit resulted in a Kd value of 5.7 μm. C, kinetic analysis of the RhoGDI interaction with unprenylated RhoA·mGDP. A typical fluorescence change observed upon mixing 0.4 μm RhoAmGDP with 8 μm RhoGDI. The inset represents a plot of the observed rates as function of RhoGDI concentration. D, fluorescence change upon mixing of 0.4 μm RhoA·mGDP·RhoGDI complex with 5 μm RhoA·GDP. E, fluorescence change resulting from mixing of 0.4 μm RhoA·mGMPPNP with 12 μm RhoGDI. The inset represents the plot of observed rates as a function of RhoGDI concentration. F, fluorescence change upon mixing of 1.2 μm RhoA·mGMPPNP·RhoGDI with 20 μm RhoA·GMPPNP.
FIGURE 2.
FIGURE 2.
Thermodynamic analysis of RhoGDI interaction with C-terminal-truncated RhoA mutants. A, the alignment of human RhoA, Cdc42, and Rac1 protein sequences. The prenylatable cysteine is highlighted in bold, the gray background represents conservative sequence of positively charged amino acid residues, and the C-terminal α-helix is underlined. The C-terminal truncation of RhoA are indicated by arrows at corresponding positions. Titration of 10 μm solution of wild-type RhoA (B) or RhoA with a C-terminal deletion of 13 residues (C) with RhoGDI.
FIGURE 3.
FIGURE 3.
Construction and analysis of prenylated fluorescent RhoA sensor. A, chemical structure of NBD-GPP in comparison with structures of FPP and GGPP. B, SDS-PAGE analysis of RhoA-GNBD eluted from Superdex 200 size exclusion column. The upper panel shows a gel stained with Coomassie Blue, whereas the lower panel shows a fluorescent scan of the same gel (excitation laser, 473 nm; cutoff filter, 510 nm). C, ESI-MS analysis of RhoA-GNBD. D, emission spectra of 50 nm solution of RhoA-GNBD before or after addition of 1 μm RhoGDI or GGTase-I. E, titration of RhoGDI to 50 nm solution of RhoA-GNBD. The fluorescence of the NBD group was excited at 479 nm, and the emission was collected at 560 nm. The Kd value of 2 nm was calculated by fitting data to the quadratic equation using Grafit 5.0. F, kinetic analysis of RhoGDI interaction with the RhoA-GNBD. The graph represents a typical time course of fluorescence signal changes upon rapid mixing of 100 nm RhoA-GNBD and 1.5 μm RhoGDI at 25 °C. The solid curve shows the single-exponential fit to the data. The inset represents a plot of the observed pseudo-first order rate constant kobs versus concentration of RhoGDI.
FIGURE 4.
FIGURE 4.
Interaction analysis of prenylated RhoA·GDP with RhoGDI. A, titration of the RhoGDI into a mixture of fluorescently labeled 100 nm RhoA-GNBD (λex/em 479/560 nm) in the absence (open diamonds) and presence of increasing concentrations of RhoA-F: 50 (open circles), 100 (filled circles), 200 (open squares), and 400 nm (filled squares). The data were fitted to a competitive model, resulting in a Kd value of 2.5 nm. B, purification of the RhoA-GG·GGTase-I complex by size exclusion chromatography and SDS-PAGE analysis of complex containing fractions. Arrows indicate elution volumes of molecular weight standards. C, the MALDI-MS analysis of the purified RhoA-GG·GGTase-I complex. D, titration of RhoGDI to a 15 nm solution of RhoA-GNBD in the absence (open triangles) or presence of 100 (open circles), 200 (filled circles), or 400 nm (open squares) RhoA-GG·GGTase-I complex. The data were fitted globally by numerical simulation to a competitive binding model in the program Dynafit 4.0.
FIGURE 5.
FIGURE 5.
Interaction analysis of prenylated RhoA interaction with RhoGDI. A, competitive displacement of the CFP-RhoA·GDP-GG·RhoGDI-TMR complex by farnesylated RhoA observed by FCCS. Open circles correspond to the value of the cross-correlation function at the indicated concentrations of RhoA-F. Solid line is the fit of experimental data to a competitive binding model, leading to a Kd value of 21 pm. Bars represent S.D. from two independent measurements. B, titration of RhoGDI into the 25 nm solution of RhoA-GNBD in the absence (filled circles) or presence of 100 (open circles), 200 (filled triangles), or 400 (open triangles), and 600 nm (filled diamonds) of the RhoA·GMPPNP-GG·GGTase-I complex. The data were fitted globally by numerical simulation to a competitive binding model in the program Dynafit 4.0. The Kd values obtained for the RhoA-GG/GGTase-I interaction is ∼12 nm, and for RhoA-GG/GMPPNP/RhoGDI, the affinity is ∼2.9 nm. C, kinetics of dissociation of the CFP-RhoA-GG·RhoGDI-citrine complex, measured by the changes in FRET efficiency (λex 436 nm/λem,DONOR 470 nm, and λem,ACCEPTOR 530 nm). In the experiment, 30 nm CFP-RhoA was displaced from the complex by the addition of 2 μm farnesylated RhoA. Purple curve corresponds to a EFRET change upon dissociation of CFP-RhoA·GDP-GG·RhoGDI-citrine complex, whereas red represents the change in EFRET of dissociating CFP-RhoA·GMPPNP-GG·RhoGDI-citrine. Blue and green curves are EFRET of corresponding complexes alone. Black solid lines are fits to a single exponential function with offset. D, represents titration of CFP-RhoAGDP-GG·RhoGDI-citrine complex with farnesylated RhoA. The fit of experimental data led to a Kd of the CFP-RhoAGDP-GG interaction with RhoGDI of 16 pm.
FIGURE 6.
FIGURE 6.
Structure of RhoA·GMPPNP-GG·RhoGDI complex (PDB 4F38). A, overall structure of the complex. RhoGDI is displayed as a gray molecular surface, whereas RhoA is displayed in ribbon representation. The nucleotide and the conjugated geranylgeranyl isoprenoid are displayed in ball-and-stick representation. The Mg2+ is displayed as a space-filling magenta ball. B, 2.5 σ (Fo − Fc) difference in electron density of the bound GMPPNP and geranylgeranylated cysteine at the RhoA C terminus before incorporation into the model. C, superimposition of the prenylated CDC42·GDP and RhoA·GPPNHP complexed with RhoGDI. D, as in C but RhoA·GMPPNP complexed with RhoGDI is superimposed with unbound GMPPNP-associated (red) and GDP-associated (blue) forms of RhoA.
Scheme 1.
Scheme 1.
Mechanism of RhoA extraction by RhoGDI.
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