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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anal Biochem. Author manuscript; available in PMC Oct 15, 2009.
Published in final edited form as:
PMCID: PMC2633595
NIHMSID: NIHMS69504
Flow Cytometry for Real-Time Measurement of Guanine Nucleotide Binding and Exchange by Ras-like GTPases
Samantha L. Schwartz,*1 Mathewos Tessema,*1,2 Tione Buranda,1 Olena Phlypenko,3 Alexey Rak,3,4 Peter C. Simons,1 Zurab Surviladze,1 Larry A. Sklar,1 and Angela Wandinger-Ness1
1Department of Pathology MSC08-4640 and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131
2Lovelace Respiratory Research Institute, Albuquerque, NM 87108-5127
3Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, 44227 Dortmund Germany
4Sanofi-Aventis, Centre de Recherche Paris, 13, Quai Jules Guesde-BP 14, 94403 Vitry sur Seine Cedex, France
Correspondence should be addressed to: Angela Wandinger-Ness, University of New Mexico HSC, 2325 Camino de Salud NE, CRF225, Albuquerque, New Mexico 87131, Phone: 505-272-1459, FAX: 505-272-4193, Email: wness/at/unm.edu
*Authors contributed equally to the work
Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Ras-family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg2+, with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases.
Keywords: Rab and Rho GTPases, membrane trafficking, actin remodeling, nucleotide binding and exchange, fluorescent GTP analogues
Small molecular weight, Ras-like GTPases regulate a variety of cellular functions ranging from signal transduction (Ras), cytoskeletal rearrangements (Rho), membrane dynamics and protein synthesis (Arf/Sar) to vesicular (Rab) and nuclear transport (Ran) [14]. Abnormal regulation of GTPases has been implicated in wide range of diseases including cancer, pigmentation and immune disorders, as well as neurological disease [58]. The structural conformation and hence the activity of GTPases is mainly regulated by the nature of the bound guanine nucleotides [9]. Binding to GTP activates the GTPase and induces a conformational change that regulates interactions with downstream effector proteins. The hydrolysis of GTP to GDP due to the intrinsic GTPase activity of the protein inactivates the GTPase, though often the intrinsic hydrolysis rates are slow [1012]. In vivo, activation and inactivation is further regulated by the binding of numerous accessory proteins that serve to enhance nucleotide exchange and hydrolysis [1, 3, 13]. Developing a convenient method that accurately measures the kinetics of nucleotide binding of GTPases is crucial for monitoring pathways controlled by these pivotal regulatory proteins. Although most GTPases share similar structural features such as switch regions and nucleotide binding pockets, the variations within these regions enable them to play unique roles in cellular function. Discerning the differences between these GTPases, as well as what conditions affect nucleotide binding, is crucial to understanding disease pathways and developing drug targets. Detailed analyses of the kinetic parameters of nucleotide binding and release are also expected to provide key mechanistic insight and suggest rate-limiting events in pathway regulation.
A variety of methods have been utilized to measure nucleotide binding to GTPases, some of which are non-quantitative and none of which allow rapid, real-time measurement as required for high through-put screening and analyses of complex mixtures or cell lysates. In the cell biology community, nucleotide binding is most often confirmed by ligand overlay blotting, TLC analysis of bound nucleotide following protein immunoprecipitation or in vivo FRAP assays [1417]. Such assays assess nucleotide binding qualitatively but cannot readily measure affinity for different nucleotides or interaction kinetics. Quantitative analyses have relied on the use of radiolabeled nucleotides and measurement via filter binding and liquid scintillation counting [17, 18] or the use of reverse-hase HPLC [11]. A highly quantitative fluorescence approach was first pioneered for the Rab GTPases and entails monitoring intrinsic tryptophan fluorescence changes upon nucleotide binding or tryptophan FRET to 2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted fluorescent nucleotides [11, 12, 19]. However, this approach is not broadly applicable to GTPases that lack the requisite tryptophan residues, including important small molecular weight GTPases such as Ha-Ras p21 and heterotrimeric G proteins among others. In general, the need for large quantities of purified protein, multi-step procedures for preparing nucleotide-free protein, specialized instrumentation, artifacts induced by filter-binding and radiolabeled analogues may explain why these techniques have not been more broadly employed in the characterization of the large number of small GTPase family members.
Recent efforts to broaden the applicability of fluorescent strategies for measuring nucleotide binding to heterotrimeric and small GTPases led to the development of a series of fluorescent BODIPY-conjugated GTP analogues and quantification of the binding and dissociation of fluorescent nucleotide analogues by spectrofluorometry [2022]. Here we describe a rapid bead-based, flow cytometry assay using BODIPY FL nucleotide analogues to measure the kinetics of nucleotide binding and dissociation from Rab7, a small GTPase and member of the Ras superfamily involved in endosome maturation and lysosome biogenesis [2325]. Rab7 was selected for these studies because its kinetic and structural properties are well described, thereby enabling comparative evaluation with established methods [11, 12, 2628]. Real time measurements with fluorescence detection by flow cytometry discriminate free GTP analogues from those bound to GTPases on a bead, and can be used for a wide range of binding conditions. We exploit this homogeneous discrimination to investigate the difference in affinity for different nucleotides, as well as to reveal structurally confirmed differences in nucleotide binding pocket structure between Rab7 and the RhoA and RhoC GTPases that are important in actin dynamics, cell-cell adhesion and trafficking [29]. The assay approach has already been shown to have further utility for protein-protein interaction studies, comparative evaluation of multiple GTPase family members and drug screens (PubChem AID # 757-61, and AID 764; http://nmmlsc.health.unm.edu/assays.shtml).
Reagents
All reagents were of analytical quality and unless otherwise noted were from Sigma (www.sigmaaldrich.com). Plasticware was from VWR (www.vwr.com). Sephadex G-25, glutathione Sepharose 4B, and Superdex peptide beads were from Amersham Biosciences (www.amershambiosciences.com); the Superdex peptide beads were 13 µm with an exclusion limit of 7,000 Da, and were extruded from a column. Purification and filter concentrators were from Millipore. Fluorescein and BODIPY® (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or dipyrromethene-boron-difluoride) nucleotide analogues (BODIPY FL GTP 2’-(or-3’)-O-(N-(2-aminoethyl)urethane, G-12411; BODIPY FL GTP-γ-NH amide, G-35778; BODIPY FL GDP 2′-(or-3′)-O-(N-(2-aminoethyl)urethane), G22360) were from Invitrogen Molecular Probes (http://probes.invitrogen.com). Concentrations for BODIPY nucleotides were determined from absorbance measurements using an extinction coefficient value of 80,000 M−1cm−1 (Invitrogen Molecular probes). GST-RhoA purified from E. coli was purchased from Cytoskeleton (http://www.cytoskeleton.com/). GST-RhoC in vitro translated in a wheat germ system was purchased from Abnova Corp. (H00000389/P01) and glutathione (GSH) was removed by spin concentration in the presence of 0.1% bovine serum albumin.
Expression and purification of GST-tagged proteins
Recombinant DNA expression was performed in accordance with NIH guidelines and with approval by the institutional biosafety office. A fusion protein between glutathione S-alkyl transferase and green fluorescent protein (GST-GFP) was constructed by inserting an EcoRI-NotI fragment from pEGFP-N2 (Becton-Dickinson Biosciences) into pGEX-6P-1 (Amersham Biosciences) as described [30]. A vector encoding GST-Rab7 was constructed by inserting PCR-amplified, wild-type, canine Rab7 into pGEX-5X-2 (Amersham Biosciences, Piscataway, NJ) using standard recombinant DNA techniques [31]. BL21 (DE3) E. coli were individually transformed with the expression vectors and grown overnight at 37°C on Luria-Bertani broth (LB)-agar plates with 100 µg/ml ampicillin. About 20 colonies were transferred into 100 ml LB broth with 100 µg/ml ampicillin and grown at 37°C to a bacterial density of 0.6 absorbance units at 595 nm. For GST-Rab7 expression, the culture was transferred to room temperature, 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce expression of the properly folded, active fusion protein and further incubated at room temperature for 16–18 h. For GST-GFP, the incubation temperature was reduced to 30°C, 0.25 mM IPTG was added and culture continued at 30°C for 4–5 h. Bacterial cells expressing the fusion proteins were then collected by centrifugation and quick frozen directly as pellets at −20°C. Pellets were then resuspended in PBS to a final volume of 10 ml and bacteria were lysed using a Branson Sonicator 250 at setting ‘3’ with 6 bursts of 10 s, followed by a 30 min incubation with the addition of 0.5 ml 10% (v/v) Triton X-100. The lysates were cleared by centrifugation at 8,000 g for 10 min. The GST-fusion proteins were subsequently affinity purified by sequential passage over two separate 0.5 ml columns of glutathione Sepharose 4B. The affinity columns were rinsed two times with 10 ml of buffer and the bound GST-fusion proteins were eluted with 10 mM GSH, incubated with the resin for 10 min at 4°C. The eluates were again passed through gel filtration columns to remove free GSH or simultaneously purified and concentrated using Millipore Amicon Ultra Tubes MW cut-off 30,000 (Catalog# UFC903024). Following the second gel filtration, the GST-fusion proteins were concentrated using separate Microcon YM-30 filters, the retentates were washed three times with fresh buffer, and the final filtrates and retentates were collected. The purity of the retentate was verified by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie staining (Fig. 1A). The concentrations of the purified fusion proteins were quantified by the method of Bradford [32] using bovine serum albumin as a standard, ethylene glycol was added to 50% by volume and aliquots were stored at −20°C until needed.
Fig. 1
Fig. 1
Purified GST-Rab7 can be immobilized on GSH beads and GTP-binding can be detected by flow cytometry using femtomoles of protein
Synthesis of GSH loaded beads (bGSH)
Flow cytometry suited, high glutathione density beads (bGSH) were prepared by loading Superdex peptide beads with GSH as previously described [30]. Briefly, Superdex peptide beads of 13 µm diameter and 7,000 Da exclusion limit were extruded from a column, activated with a water-soluble bis-epoxide, and then coupled to glutathione. One ml of a 50% slurry Superdex peptide beads in 20% ethanol was reduced to a wet cake using a 15 ml coarse sintered glass filter, and washed three times with 15 ml water to remove the ethanol. The wet cake was transferred to a small screw-cap tube, and the filter was rinsed with 0.3 ml of water. The beads were suspended by vortexing, then 60 µl of 10 M NaOH and 300 µl of 1,4-butanediyl diglycidyl ether were added, and the suspension was rocked gently at 40°C for 4 h. The epoxy-activated beads were rinsed four times on the filter and 600 µl of 100 mM GSH in 100 mM sodium phosphate, pH re-adjusted to 7.5, 1 mM EDTA, was added. The beads were kept in suspension for 16 h at 40°C, and then rinsed twice with 0.01% dodecyl maltoside. The remaining active sites were blocked with 1% 2-merca toethanol for 2 h. The beads were rinsed twice and stored in HPS (30 mM HEPES, pH 7.5, 100 mM KCl, 20 mM NaCl, 0.01% dodecyl maltoside) containing 1 mM MgCl2 and 0.02% NaN3 at 4°C as a 50% slurry, which corresponds to ~2.5 × 105 high GSH density beads/µl, or 25 assays of 104 beads/10 µl.
Flow cytometry measurements and molecular assembly of GST-Rab7 and GST-RhoA proteins on GSH beads
All binding and measurements were performed in the presence of HPS buffer containing 1 mM EDTA or 1 mM MgCl2 as noted. A Becton-Dickinson FACScan flow cytometer (Sunnyvale, CA, USA) with a 488 nm excitation laser and standard detection optics was used for all assays as previously described [33]. GST-proteins (1µM, GST-Rab7 or GST-RhoA), were incubated in 96-well plates at 4°C, overnight with 103 glutathione beads/µl in a total volume of 10 µl HPS buffer with 1 mM DTT (dithiothreitol/ Cleland’s reagent) added fresh and 1 mM EDTA or 1 mM MgCl2 as noted. Unbound protein was removed by centrifugation and beads were resuspended in fresh buffer, the wash step repeated once before storage at 4°C until needed. We have previously shown that GST-protein assemblies on GSH beads are very stable [30] and disassemble from the glutathione beads, at very slow rates 1.3 ×10−4 s−1 (or t1/2=1.7 hr) only when highly diluted to limit rebinding. Thus, the bead bound protein is known to be sufficiently stable to enable the study of secondary interactions between GST-proteins and fluorescently tagged guanine nucleotides with minimal loss of the “foundation” assembly.
Dose dependence binding assays
For all dose dependence assays, 104 beads loaded with GST-protein were incubated for 2 h at 4°C with varying concentrations of fluorescent guanine nucleotide (0–2 µM) in a total volume of 10 µl on a 96-well plate. For measurement, samples were transferred to a tube suitable for flow cytometry and diluted at least 10-fold in the appropriate HPS buffer. This dilution step is necessary to optimize the discrimination of bead-associated fluorescence and background fluorescence of soluble proteins, and also ensures sufficient sample volume for measurement.
Competitive binding assays
For the competition assays, 104 beads loaded with GST-protein were incubated for 2 h at 4°C with 100 nM fluorescent nucleotide and the appropriate concentration of non-fluorescent nucleotide competitor in a 96-well plate with a total volume of 10 µl. Measurements were performed as described above.
Kinetic assays
Association measurements were performed by incubation of beads loaded with GST-protein (103 beads/µl) at 4°C with the appropriate concentration of fluorescent nucleotide in a 1.5 ml tube. At the desired time points 10 µl aliquots were removed and measured on the flow cytometer. Dissociation was measured by removing a 10 µl aliquot of equilibrated, fluorescent nucleotide-bound protein on beads and diluting it 10-fold in the presence of 1 mM non-fluorescent GTP. In addition, real time measurements of changes in fluorescence were taken using the kinetic mode of the cytometer.
Standardization of flow cytometry data with fluorescence calibration beads
We used standard fluorescence calibration beads to account for day-to-day variation in bead fluorescence, changes in detector settings and use of different flow cytometers [3436]. The standard calibration beads used here were commercial standards from Bangs Labs (www.bangslabs.com) Quantum™ FITC MESF beads. These beads are optimized for fluorescein but can be used for any fluorophore being analyzed under the same excitation and detection optics as long as a suitable correction factor is applied to account for the spectroscopic differences between the fluorescein and target fluorophore (e.g. BODIPY FL) [37]. The calibration scheme has been recently described in detail [37]. Given that the measured fluorescence intensity of any given molecule is proportional to, I0εexϕ%T; where I0 is the intensity of the light source, εex is the absorption coefficient of the fluorophore at the excitation wavelength and ϕ is the quantum yield of the fluorophore and %T is the percent fraction of fluorescence light transmitted by the 530 nm, 30 nm wide bandpass filter (upto 80% maximum transmittance) and is used to account for the spectral mismatch between the sample fluorophore and the fluorescein standard. Therefore, to apply Quantum FITC MESF beads to BODIPY labeled assemblies we derived a correction factor based on the ratio using values for unquenched fluorophores [37]:
equation M1
(1)
Thus, the measured BODIPY intensity is attenuated by a factor of 0.36. The parameter that most significantly affects the measured output of the BODIPY fluorophore is the off-resonance excitation (55% of its λmax) of the fluorophore compared to fluorescein, which is excited at its absorption maximum.
Data Analysis
Multiple trials were used to generate means and standard errors. For detailed fitting parameters see Table 1 and Table 2. Curve fitting and analyses utilized GraphPad Prism software (www.graphpad.com).
Table 1
Table 1
Summary of GST-Rab7 binding to BODIPY nucleotide analogs
Table 2
Table 2
Comparison of GST-Rab7 and GST-RhoA binding to BODIPY-GTP analogs
Purified GTPases can be immobilized on GSH beads and GTP-binding can be detected by flow cytometry
We previously demonstrated the utility of flow cytometry for accurately measuring the dynamics of interaction between GST-GFP fusion proteins and glutathione beads [30]. Here we extend the utility of glutathione beads to the characterization of the equilibrium binding and association and dissociation kinetics between GST-Rab7 and BODIPY FL tagged guanine nucleotides. The molecular assembly schemes are shown in Equation 2 and Equation 3. The first step entails binding of purified GST-Rab7 (Fig. 1A) to the GSH beads (bGSH) (step 1 in Fig. 1B), and is expected to have characteristics similar to the previously described GST-GFP [30]. The second step is of biological interest and involves binding of the Rab7 protein to the fluorescently labeled nucleotide (BODIPY FL GTP) (Step2 in Fig. 1B).
equation M2
(2)
equation M3
(3)
The strong affinity interaction between glutathione beads and GST-proteins was documented in the chromatography literature over three decades ago [38]. Here, we examined the binding between glutathione beads and GST-Rab7 solely to establish quantitative limits on the number of binding sites per bead under our assay conditions. GST-Rab7 pre-incubated with saturating concentrations (10 µM, see Fig. 2C) of BODIPY FL GTP was used to determine the maximum number of GST-Rab7 occupied sites. Fig. 1C shows a parabolic plot of GTP sites/bead versus concentration of GST-Rab7 (bound to BODIPY FL GTP). The sites/bead were determined using calibration beads as described in the methods section. At the maximal concentration of protein, a proximately 5×106 GST-Rab7 molecules bound to each bead. This represents a concentration of 2 nM bead-bound protein and translates to ~ 20 femtomoles of protein per 10 µl reaction. The dose dependence of GST-Rab-BODIPY FL GTP binding to the GSH beads revealed an apparent dissociation constant of Kd = 190 nM (Fig. 1C). An alternative analysis of the apparent affinity of GST-Rab7 for the GSH beads was based on competing the non-fluorescent protein with fluorescent GST-GFP protein. The inhibition constant Ki = 107 nM for GST-Rab7 was com arable to the Kd value determined in Fig. 1C. It should be noted that the protein-bead binding constants were not obtained under true equilibrium conditions, which requires long term incubation at 4°C. Therefore, the Kd values are principally useful as quantitative parameters for setting up the assay platform rather than being thermodynamically meaningful. [39]. On the other hand, protein saturated beads can be generated in a relatively short time when micromolar concentrations of GST-proteins are used.
Fig. 2
Fig. 2
Kinetic measurement of nucleotide affinity and Mg2+ dependence
Effect of magnesium on the equilibrium exchange of endogenous for exogenous GDP in the nucleotide binding pocket
Mg2+ ions are believed to play a significant role in the stabilization of nucleotide binding, to Ras, Rho and Rab GTPases in addition to acting as cofactors in nucleotide hydrolysis [18, 4042]. The nucleotide-binding pocket of purified GTPases is usually occupied with endogenous GDP. Therefore, the binding of fluorescently tagged nucleotides to Rab7 GTPase is expected to involve an exchange of the endogenous GDP. To enhance the probability of nucleotide-exchange we examined the effects of incubating exogenous nucleotides with Rab7 GTPase loaded beads in the presence of EDTA. Fig. 2A shows the differences in exchange of endogenous GDP for fluorescently labeled GTP and affinity for BODIPY FL GTP [18, 41] in Mg2+-containing or -depleted buffers. The stabilizing influence of Mg2+ on the interaction of endogenous GDP with the Rab7 nucleotide binding pocket is a parent in the parabolic plot of exchangeable sites of BODIPY FL GTP versus concentration of the titrated exogenous nucleotide. In the presence of Mg2+, BODIPY FL GTP binds with higher affinity Kd = 20 nM to relatively fewer exchangeable sites, whereas depletion of Mg2+ results in a higher number of exchangeable sites of diminished affinity (Kd =150). The reduced affinity is consistent with an increase in the dissociation rate of the nucleotide in the absence of Mg2+ as expected. An analysis of the early kinetics of dissociation of BODIPY FL GTP from Rab7 when competed by a large excess of unlabeled GDP showed the dissociation rate to be 7 times faster in the absence of Mg2+ relative to when Mg2+ was present (Fig. 2B). The latter result is consistent with the 8-fold decrease in affinity of the nucleotide interaction with Rab7 in the absence of Mg2+ (Fig. 2A), demonstrating that accurate kinetic measurements of Rab7 nucleotide binding are possible in both the presence and absence of magnesium cations.
To convincingly demonstrate that under our assay conditions the binding of BODIPY FL GTP to the Rab7 nucleotide-binding pocket largely involves an exchange of endogenous GDP [11, 18], we examined the association kinetics of BODIPY FL GTP with Rab7 beads by varying the concentration of ligand over a 10-fold range in Mg2+-depleted buffer. The association data are shown in Fig. 2C. Because the ligand concentration remains constant throughout the experiment, the data were analyzed in terms of pseudo-first order kinetics. The on-rates (t1/2) as a function of concentration were then plotted as shown in Fig. 2D. Because the association-rates are constant irrespective of a 10-fold change in ligand concentration, we conclude that the on-rates are indeed governed by the rate of nucleotide exchange. Therefore, in situations where the intrinsic association rate is of interest care must be taken to remove all endogenous nucleotide as previously described [11, 41, 42].
BODIPY FL GDP binds to the nucleotide binding pocket more tightly than GTP analogues
We used 3 different experimental measures of the binding affinity of Rab7 pocket for the fluorescently tagged GDP and GTP analogues. Fig. 3A shows a parabolic plot of bound nucleotide analogues versus concentration. The data show that BODIDY FL GDP binds to the nucleotide binding pocket with a 3-fold higher affinity (Kd = 54 nM) compared to BODIDY FL GTP (Kd = 150 nM). This difference in affinity is well correlated to a 3-fold faster dissociation of GTP (koff = 7.0 × 10−3 s−1) from the Rab7 compared to the GDP (koff = 2.3 × 10−3 s−1) shown in Fig. 3B. We also analyzed the competitive displacement of BODIDY FL GTP from Rab7 using GDP versus GTPγS, a nonhydrolyzable analogue of GTP (Fig. 3C). The resulting inhibition measured for GDP, (Ki = 13 nM) and and GTPγS, (Ki= 40 nM) further corroborated the 3-fold higher affinity of Rab7 for GDP relative to GTP.
Fig. 3
Fig. 3
GST-Rab7 has different affinities for BODIPY FL GTP and BODIPY FL GDP
Fluorescent nucleotide analogues reveal structure/function differences between GTPases
Because BODIPY FL GTP is subject to hydrolysis, the non-hydrolyzable, BODIPY FL GTP-γ-NH analog is frequently used. BODIPY FL GTP-γ-NH differs from BODIPY FL GTP in that the fluorophore is conjugated to the nucleotide via the γ-hosphate as opposed to the 2’ or 3’ oxygen of the ribose ring (Fig. 4A) [20]. Remarkably, GST-Rab7 showed 90% lower binding affinity for the BODIPY FL GTP-γ-NH relative to the BODIPY FL GTP (Fig. 4B). In the presence of Mg2+, GST-Rab7 yielded a maximal binding Bmax= 60 × 103 fluorophores/bead for BODIPY FL GTP-γ-NH compared with a maximal binding Bmax= 650 × 103 fluorophores/bead for BODIPY FL GTP.
Fig. 4
Fig. 4
Rab7 discriminates BODIPY FL GTP nucleotide analogues conjugated via theribose or at the γ-phosphate position
To determine whether other small molecular weight GTPases were equally sensitive to the structural modification of nucleotides we also carried out similar binding experiments using GST-RhoA. In contrast to GST-Rab7, GST-RhoA, showed no difference in nucleotide analogue binding (Fig. 4B), signifying that the nucleotide analogue preference may be GTPase specific. The dose response curve for GST-RhoA binding to BODIPY FL GTP yielded a maximal binding Bmax= 700 × 103 fluorophores/bead and a Kd = 80 nM (Fig. 4B).
The unique inability of GST-Rab7 to bind the BODIPY FL GTP-γ-NH nucleotide prompted a structural comparison. The crystal structures for both GTP-bound Rab7 and RhoA reveal how placement of the fluorophore group could lead to such a drastic change in binding affinity (Fig. 4C). The ribose and phosphate groups make distinct contacts within the Rab7 and RhoA nucleotide binding pockets. Furthermore, the Rab nucleotide binding pocket leaves the ribose ring of the nucleotide solvent exposed, whereas the γ-hosphate is buried within the protein. A bulky BODIPY FL group on the γ-hosphate may not allow the nucleotide to be accommodated within the binding pocket of Rab7. In RhoA the binding pocket leaves both the ribose and the γ-phosphate solvent exposed so either BODIPY FL or BODIPY FL GTP-γ-NH can fit into the pocket. Analogous results were found for RhoC (not shown).
We report a new bead based flow cytometry assay to measure in real time, the affinity and kinetics of nucleotide binding to small GTPases, using GST-fusions of Rab7 and RhoA as test cases. The particular features of the assay that assure broad utility are the facts that: (1) it circumvents the need for radiolabeled analogues; (2) only femtomoles of purified protein are needed per assay; (3) the assay may be run on a conventional flow cytometer; (4) the assay is quick and measures binding in real time without large numbers of wash steps; and (5) it may be run with or without magnesium.
The assay enables the sensitive identification of comparative binding differences between GTPases and their nucleotides, though as executed here does not establish the absolute kinetic and affinity constants for the GTPase. The nucleotide binding pocket of GTPases is nearly always occupied by GDP except in association with molecular chaperones that promote exchange and stabilize the nucleotide free form [19, 43, 44]. Consequently, considerable effort must be taken to purify small GTPases free of nucleotide and magnesium as described by Simon et al. [11]. Since the procedure of stripping nucleotide is laborious and accompanied by loss of activity, it is advisable only in situations where it is critical to determine absolute constants. The dissociation constant and kinetic rates determined by flow cytometry are not directly comparable to those determined in nucleotide free systems where a Kd = 6 nM for Rab7 binding to methylanthraniloyl (mant) GTP was reported [11]. Instead, our values agree well with those measured under conditions allowing nucleotide exchange for BODIPY FL analogues and using fluorescence spectroscopy [21]; in which case the nucleotide binding affinities (Kd) for Cdc42, Rac1, RhoA, and Ras ranged between =10–100 nM. The use of a reduced Mg2+ system diverges from the normal in vivo environment that GTPases reside in. Mg2+ and Mn2+ are important for securing the nucleotide into the binding pocket and Mg2+ drastically slows nucleotide off rates [41]. Because the flow cytometry based measurement reflects exchange of endogenous nucleotide for fluorescently labeled BODIPY FL GTP, we found it useful to remove Mg2+ ions, which tend to limit this exchange (c.f. Fig. 2). Nevertheless, as shown here, the applicability of our assays is independent of Mg2+, but can be used when desired or necessary.
The GST-Rab7 binding affinities for several nucleotides and nucleotide analogues yielded three interesting results. First, although Rab7 has a 3-fold higher affinity for GDP Kd = 54 nM than for GTP Kd = 150 nM, however, this is a relatively small difference considering that the cell and tissue concentration of GTP is in the micromolar range (19–276 µM with erythrocytes having the most variable levels) and 2- to 9-fold higher than GDP [4549]. Thus, in the cellular context it makes sense that the nucleotide bound state must be regulated by accessory proteins to preclude purely concentration dependent binding. Second, recent work by Korlach et al. [21] suggests that BODIPY FL nucleotides may act analogous to guanine nucleotide exchange factors (GEFs) and aid displacement of endogenous, unlabeled nucleotide. However, because the bound BODIPY FL nucleotides were completely competed off with unlabeled nucleotides we suggest that the BODIPY moiety is not a major factor in nucleotide exchange or binding. Third, Rab7 is sensitive to substitution at the γ-phosphate position. A recent paper by Zhang et al. suggests that during nucleotide exchange on Ras the incoming nucleotide (GTP) attacks the nucleotide binding pocket of Ras through its β- and γ-phosphate groups to displace GDP [43]. The observed sensitivity of GST-Rab7 to changes in the γ-phosphate region of the nucleotide may imply a conserved mechanism for nucleotide exchange among the Ras-related GTPases.
Unlike many other GTPases, GST-Rab7 exhibited a marked inability to bind BODIPY FL GTP when the fluorophore was attached to the γ-phosphate as in BODIPY FL GTP-γ-NH. Relative to RhoA, we find that GST-Rab7 exhibits a 90% decrease in binding affinity to BODIPY FL GTP- γ-NH, while GST-RhoA has no preference. This result is nicely explained by structural differences in the nucleotide binding pockets of the two GTPases with Rab7.
GTPases play a crucial role in regulation of a multitude of cellular functions. An understanding of the specific differences between GTPases, and their nucleotide binding will become increasingly important as we try to gain insight into mechanisms of disease. We have presented a rapid and sensitive assay for detection of nucleotide binding dynamics. With only femtomoles of protein required for individual measurements, and without complicated purification steps this assay approach can be used to quickly investigate and compare nucleotide binding of individual mutant GTPases, make comparisons of GTPases involved on the same pathway, and using modified beads for high throughput screening, identify effector molecules and or small molecule inhibitors and activators (PubChem AID # 757–61, and AID 764; http://nmmlsc.health.unm.edu/assays.shtml).
Acknowledgements
This work is generously supported by the National Science Foundation MCB0446179 and R03MH081231 to AWN, K25AI60036 to TB, and CA1181000 and MH074425 to LAS. MT was supported by a UNM Research and Allocations (RAC) postdoctoral fellow award. OP and AR are supported by European Young Investigator Award to AR, see www.esf.org/euryi. We gratefully acknowledge Ms. Elsa Romero, Mr. Jacob Agola and Mr. Mark Carter for technical assistance and Ms. Janet Kelly for administrative support. DNA Research Services and KUGR-UNM Genomics Resource provided expert assistance and instrumentation for DNA sequencing and Nanodrop quantification. All flow cytometry was conducted in the Cancer Research and Treatment Center Flow Cytometry Resource.
Footnotes
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