Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.
Background: The GEFs Rabin8 and GRAB are activators of the vesicular trafficking regulator Rab8.
Result: The catalytic mechanism of Rabin8/GRAB in Rab8 has been elucidated in biophysical and structural detail.
Conclusion: Rabin8 and GRAB are catalytically moderately efficient enzymes and act by disturbing Mg 2+ binding and Rab8-guanine base interactions.
Significance: Obtaining snapshots of the nucleotide exchange reaction is crucial to understanding the mechanism of Rab GEFs.
One of the hallmarks of eukaryotic cells is the intracellular movement of vesicles that transport material and allow communication between cellular compartments. The spatial and temporal regulation of vesicular trafficking is achieved by proteins of the Rab subfamily of small GTPases. Rab proteins are molecular switches and cycle between inactive GDP-bound and active GTP-bound states. When inactive, Rab proteins exist in the cytosol in complex with the GDP dissociation inhibitor but are localized to a distinct membrane when in the active state. To exert their function, Rab proteins need to be activated by a process requiring guanine nucleotide exchange factors (GEFs).6 These enzymes accelerate GDP release from and allow the binding of GTP to a Rab protein. Rab proteins can interact with effector proteins that preferentially bind the active GTP but not the GDP state. GTPase-activating proteins stimulate the very low intrinsic GTPase activity of Rab proteins and thus convert them back into the inactive form.
The Rab subfamily consists of ∼60 members in humans, and each family member has a specific intracellular localization. The correct activation of a certain Rab requires the action of a cognate Rab GEF at the proper location and at the appropriate time. Consequently, GEFs have evolved to have mechanisms that guarantee their correct membrane targeting as well as the specific recognition of one distinct Rab protein over structurally and sequentially similar family members.
The Rab protein Rab8 is involved in events such as the delivery of secretory vesicles to the plasma membrane and polarized membrane transport in epithelial cells. Rab8 also regulates cell shape, and interaction with its GEF Rabin8 appears to be crucial to this function. Rabin8 and Rab8 appear to be important in cilium formation by acting in concert with the Bardet-Biedl syndrome complex. Rabin8 is a 460-amino acid protein that consists of several domains, only two of which are functionally characterized. Rabin8 contains a central Sec2 domain with GEF activity toward Rab8. Amino acid sequence and structure comparison with the yeast homolog Sec2 predicts that the central domain of Rabin8 consists of a homodimeric parallel coiled coil. Rabin8 is thought to be recruited to its target location by active Rab11, which interacts with a C-terminal Rab11 effector domain. Another factor possessing a Sec2-like GEF domain is the 382-amino acid protein GRAB. Despite a high sequence homology of the Sec2 domain of GRAB to Rabin8, it has previously been reported that GRAB is a GEF for Rab3A rather than Rab8. However, a recent study that analyzed the activity profiles of various Rab GEFs (with a focus on the DENN domain family, which is structurally unrelated to GRAB) indicated that GRAB has GEF activity toward Rab8 but not Rab3.
The mode of action of GTPases involved in signal transduction or regulation includes activation of GDP release catalyzed by a specific GEF in almost all cases known. The basic mechanistic feature is the weakening of the otherwise very tight binding of GDP (K d values in the nanomolar to picomolar range) by interaction with GEFs, which also bind with similarly high affinity to their cognate GTPases. The effect is both thermodynamic and kinetic, implying the formation of a ternary complex between GEF, GTPase, and GDP, with a dramatic reduction in the affinities of both GEF and GDP in the ternary complex in comparison with the respective binary complexes. Several GTPase-GEF interactions have been examined thoroughly at the kinetic level, and a large number of GTPase·GEF complexes have been characterized structurally.
In this work, we have examined the interaction of the Ras superfamily protein Rab8 with two structurally highly similar GEF molecules (Rabin8 and GRAB) by kinetic and structural methods. GRAB is a GEF for Rab8 with almost identical structural properties to Rabin8. The work presented led to the identification of several intermediates in the overall GDP/GTP exchange of Rab8 in the presence of Rabin8.
Human Rab8a(1–184) and Rab8a(6–176) were expressed in Escherichia coli and purified as described previously. Expression and purification of Rab3 were performed as described. The protein-encoding sequences of full-length GRAB and the coiled-coil domains of Rabin8/GRAB were cloned into a modified pET19 vector containing an N-terminal His 6 tag followed by a tobacco etch virus protease cleavage sequence. In the case of GRAB, a synthetic codon-optimized gene was used. GRAB and Rabin8 variants were expressed in E. coli BL21(DE3)RIL by induction with 0.5 m m isopropyl β-d-thiogalactopyranoside at 20 °C for 18 h and purified by nickel-nitrilotriacetic acid affinity chromatography. After removal of the His 6 tag with tobacco etch virus protease, the GRAB and Rabin8 variants were further purified by nickel-nitrilotriacetic acid affinity chromatography followed by size exclusion chromatography. Preparative loading of Rab8a(6–176) with GppNHp was performed as described previously. To obtain a nucleotide-free Rab8a(1–184)·Rabin8(157–232) complex, Rab8·GDP was mixed with Rabin8 at 1:4 molar ratio in buffer containing 20 m m HEPES (pH 7.5), 50 m m NaCl, 150 m m (NH 4)2 SO 4, 50 μ m ZnCl 2, and 3 m m DTT. Alkaline phosphatase was added to hydrolyze GDP, and the mixture was incubated for 10 h at 4 °C. GDP hydrolysis was monitored by reversed phase HPLC, and Rab8·Rabin8 was purified by size exclusion chromatography with buffer containing 25 m m HEPES (pH 7.5), 40 m m NaCl, and 5 m m DTT after GDP was completely hydrolyzed.
All crystals in these studies were obtained by mixing 1 μl of protein and 1 μl of reservoir solution at 20 °C using the hanging drop approach. Crystals of Rab8a(6–176)·GDP were obtained by mixing the protein (20 mg/ml; buffer containing 25 m m HEPES (pH 7.5), 40 m m NaCl, 1 m m MgCl 2, 10 μ m GDP, and 5 m m β-mercaptoethanol) with a reservoir solution consisting of 16% (w/v) PEG 4000, 0.1 m CaAc 2, and 0.1 m HEPES (pH 7.0). The crystal was protected with cryo solution containing 30% (w/v) PEG 4000, 0.1 m CaAc 2, and 0.1 m HEPES (pH 7.0) before data collection. Rab8a(6–176)·GppNHp crystals (15 mg/ml; buffer containing 25 m m HEPES (pH 7.5), 40 m m NaCl, 1 m m MgCl 2, 10 μ m GppNHp, and 5 m m β-mercaptoethanol) were produced in 15% (w/v) PEG 8000, 7.5% (v/v) 2-methyl-2,4-pentanediol, and 0.1 m HEPES (pH 6.8). Nucleotide-free Rab8a(1–184)·Rabin8(157–232) (10 mg/ml)) was crystallized in 18% (w/v) PEG 3350, 0.1 m Li 2 SO 4, and 0.1 m MES (pH 6.6). Before data collection, the complex crystal was protected with cryo solution containing 30% (w/v) PEG 3350, 0.1 m Li 2 SO 4, and 0.1 m MES (pH 6.6). To produce nucleotide-bound forms of Rab8·Rabin8 complexes, the nucleotide-free Rab8·Rabin8 crystals were soaked with cryo solution containing 30% (w/v) PEG 3350, 0.1 m Li 2 SO 4, and 0.1 m MES (pH 6.6) and the respective nucleotide GDP/GTP (1 m m) for 1 h at 4 °C. Rab8·GRAB (10 mg/ml) was crystallized in solution containing 1.6 m ammonium sulfate and 0.1 m sodium acetate (pH 5.3). The crystals were protected with cryo solution containing 20% glycerol in the reservoir solution before data collection. All diffraction data were collected at 100 K at beamline X10SA of the Swiss Light Source (Villigen, Switzerland). Data were processed with XDS. The structure was determined by molecular replacement with PHASER of the CCP4 suite using Sec4 in the case of Rab8·GDP/GppNHp and Sec2·Sec4 in the case of Rab8·Rabin8/GRAB complexes as a search model. The model was then corrected by alternating rounds of refinement in REFMAC5 and manual adjustment in Coot. The nucleotide was added in the final rounds of refinement. Full data collection and refinement statistics are summarized in supplemental Table S1.
Fluorescence measurements were carried out at 25 °C in buffer containing 50 m m HEPES (pH 7.5), 50 m m NaCl, 5 m m MgCl 2, and 5 m m dithioerythritol. Rab8a was loaded with the fluorescent GDP/GTP derivatives (N-methylanthraniloyl (mant)-GDP/mant-GppNHp) as described for Sec4. The fluorescence of mant was excited at 365 nm and detected using a 420-nm cutoff filter in an Applied Photophysics stopped-flow apparatus.
Using human Rab8(6–176), we investigated the GEF properties of Rabin8 and GRAB in detail. The displacement of a fluorescent GDP analog (mant-GDP) from its complex with Rab8 catalyzed by Rabin8(153–237) in the presence of excess GDP or GTP (see discussion below concerning the choice of this fragment) showed a marked acceleration with respect to the intrinsic dissociation rate (Fig. 1A). A similar effect was seen when Rabin8 was replaced by GRAB(73–154) or full-length GRAB. The difference between the observed rate constants of Rabin8(153–237) (k obs = 0.171 s−1) and GRAB(73–154) (k obs = 0.207 s−1) is ∼20% at 10 μ m GEF. The small differences in the k obs values are not enough to exclude the possibility that they arise from errors in the determination of concentrations the GEF domains of GRAB and Rabin8. Thus, we concluded that the catalytic activities of the two GEFs are similar, and more detailed kinetic investigations were carried out with the GEF domain of GRAB. Interestingly, no acceleration of mant-GDP release from Rab3a could be observed even at an elevated GRAB concentration (10 μ m) (Fig. 1A). We concluded from this that GRAB is actually a GEF for Rab8 rather than Rab3a, at least under in vitro conditions. A similar observation has been made recently by Yoshimura et al.
FIGURE 1Kinetic analysis of the Rab8-GRAB interaction.A, comparison of full-length GRAB (GRAB fl) GEF activity with the GRAB and Rabin8 GEF domains (10 μ m) interacting with Rab8·mant-GDP (mGDP) in the presence of 100 μ m GDP. Rab3·mant-GDP did not interact with full-length GRAB (10 μ m). rel., relative; a. U., arbitrary units. B, dependence of the observed rate constant of mant-GDP/mant-GppNHp release from Rab8 on the concentration of GRAB. Plots of the obse
