©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of Guanine Nucleotide Binding on the Intrinsic Tryptophan Fluorescence Properties of Rab5 (*)

(Received for publication, April 17, 1995; and in revised form, May 24, 1995)

Julie Y. Pan Jack C. Sanford Marianne Wessling-Resnick (§)

From the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To gain further insight into structural elements involved in Rab5 function, differences in the intrinsic tryptophan fluorescence of the GDP- and guanosine 5`-O-(3-thiotriphosphate) (GTPS)-bound forms of the protein were examined. When excited at 290 nm, Rab5 displays emission maxima at 339.7 nm for the GDP-bound and 336.7 nm for the GTPS-bound forms. The tryptophan fluorescence intensity is quenched by 25% in the GTPS-bound form relative to the GDP-bound conformation. Variant Rab5 molecules were created by site-directed mutagenesis to convert the protein's two tryptophans to phenylalanine residues. Fluorescence studies reveal that the observed changes upon GDP/GTPS exchange are due to a blue shift in the emission spectra for both Trp (342.0 to 339.5 nm) and Trp (335.3 to 333.7 nm) and fluorescence quenching of Trp. Consistent with the blue shift in the emission spectra, both tryptophans are more resistant to oxidation by N-bromosuccinimide in the GTPS-bound state. These data indicate that both of Rab5's tryptophans are brought into a more sequestered, hydrophobic environment upon conformational changes promoted by guanine nucleotide exchange. Since Trp lies adjacent to Rab5's cognate switch II domain, local conformational changes would be predicted based on the known structure of Ras. However, Trp lies within a region of Rab5 potentially related to the switch III domain unique to heterotrimeric G. Thus, changes in the fluorescence properties of Trp upon guanine nucleotide exchange suggest that Rab proteins may have structure-function relationships similar to those described for heterotrimeric GTP-binding proteins.


INTRODUCTION

Rab proteins are a family of Ras-like small molecular weight GTP-binding proteins localized to distinct subcellular compartments (1, 2, 3, 4, 5, 6) . Rabs have been shown to regulate specific steps of intracellular membrane trafficking(7, 8, 9, 10, 11, 12) . One member of the Rab family, Rab5, is localized on plasma membrane, clathrin-coated vesicles, and early endosomes(2, 9, 10) . Even though it has been shown to play an important role in early events of endocytosis(9, 10, 11, 12) , the exact mechanism of Rab5 function remains to be determined. The current model for the GTPase cycle of Rab5 is that a GDP dissociation inhibitor (GDI) (^1)delivers the GDP-bound protein to target membranes(13, 14) , where a guanine nucleotide exchange factor catalyzes GDP/GTP exchange(14) . After GTP hydrolysis, GDP-bound Rab5 is retrieved from the membrane by GDI(13, 15) . Thus, membrane association and dissociation is correlated with the GTP- and GDP-bound states, respectively.

The transition between GDP- and GTP-binding states is a universal molecular switch adopted by many GTP-binding proteins to regulate a diverse set of biological functions. An understanding of the structural differences between these guanine nucleotide binding conformations can help define the basis for interactions with regulatory molecules like GDI and guanine nucleotide exchange factor. Comparison of the two guanine nucleotide-bound conformations of Ras and EF-Tu indicates significant structural changes in two regions: switch I (residues 30-38 in Ras and residues 41-62 in EF-Tu) and switch II (residues 60-76 in Ras and residues 84-96 in EF-Tu)(16, 17) . Both domains are on the surface of Ras and EF-Tu and are proposed to interact with the molecules' accessory proteins. In contrast, studies on G revealed three switch regions. Besides counterparts of switch I (residues 173-183) and switch II (residues 195-215), a third region or switch III (residues 227-238) was identified to undergo marked structural rearrangements and is thought to be unique to heterotrimeric GTP-binding proteins(18) .

Based on this crystallographic information, intrinsic tryptophan fluorescence measurements have proven to be a sensitive means to detect local conformational changes in the switch regions of G(19, 20) , G(21) , and Ras(22, 23) . G and G both contain two tryptophans, one of which resides in the molecules' switch II domain. The fact that fluorescence properties of G and G are sensitive to GDP/GTP exchange(19, 20, 21) indicates that these residues can detect conformational changes within this domain. In contrast, native Ras does not contain any tryptophan residues, but three altered forms have been studied with tryptophans introduced at position 28 (immediately upstream of the switch I domain), position 56 (four amino acids upstream of the switch II domain), or position 64 (within the switch II domain); each of these Ras variants displays fluorescence properties sensitive to GDP/GTP exchange(22, 23) .

Since crystallographic information is not yet available for Rab5, we have examined the intrinsic tryptophan fluorescence of the protein to detect structural changes promoted by guanine nucleotide exchange. Sequence alignment of Rab5, Ras, and heterotrimeric G shown in Fig. 1indicates that Trp is located immediately adjacent to the region that is cognate to the switch II of Ras (16) and G(18) , whereas Trp is within a domain potentially related to the switch III region of G(18) . Thus, conformational rearrangement in both regions of Rab5 should be reported by Trp and Trp, respectively. Our characterization of the intrinsic tryptophan fluorescence properties of Rab5 and two altered forms with Trp Phe substitutions suggests that the nature of conformational changes promoted by GDP/GTP exchange may be similar to features observed for heterotrimeric GTP-binding proteins.


Figure 1: Sequence alignment of Ras, G, and Rab5 in the switch II and III regions. Protein sequences of Rab5, H-Ras, and G were aligned using the GCG package, version 7.3 (Genetics Computer Group, Madison, WI); shown is the region adjacent to the highly conserved phosphoryl-binding domain present in all GTP-binding proteins as indicated. Double and single dots represent identical and semi-conserved amino acids, respectively, determined based on identities tabulated by Dayhoff et al.(24) . The switch II domains of Ras and G and the switch III region of G are highlighted. The highlighted regions in Rab5 represent the L4/alpha2/L5 (predicted fourth loop, second alpha-helix, fifth loop as switch II) and alpha3/L7 (predicted third alpha-helix, seventh loop as switch III) domains, adopting the nomenclature of Stenmark et al. (29) .




EXPERIMENTAL PROCEDURES

Expression and Purification of Recombinant Rab5 and Trp Phe Variants

The cDNA for Rab5, a kind gift of Dr. A. Tavitian (1) , was subcloned into M13 to construct two Trp Phe variants of Rab5, Rab5 and Rab5, by site-directed mutagenesis using the method of Kunkel et al. (25) exactly as described previously (11) . This was accomplished using the oligonucleotides 5`-CAGCTGTATCAAAGATTTCAAAC-3` and 5`-GTTCTTTAACAAAATTTTTTGCT-3`, respectively. Rab5 cDNAs were inserted into a pT7.7 expression plasmid by directional subcloning as described(11) , and DNA sequences were confirmed by the dideoxy chain termination method of Sanger et al. (26). Plasmids were transformed into BL21(DE3) Escherichia coli cells (Novagen) by electroporation, cultures were grown to log phase in TB (1.2% bacto-tryptone, 2.4% bacto-yeast extract, 0.4% glycerol, 0.17 M KH(2)PO(4), and 0.72 M K(2)HPO(4)) containing 50 µg/ml carbenicillin, and expression of protein was induced upon addition of 0.8 mM isopropyl beta-D-thiogalactoside. After 2.5 h, cells were collected by centrifugation, resuspended in 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 µg/ml lysozyme, 1% CHAPS, and sonicated. The cell lysate was centrifuged at 16,000 times g at 4 °C for 10 min, and the supernatant was added to volume of DEAE-cellulose (Whatman) preswollen in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 mM MgCl(2), 0.1% CHAPS, 1 µM GDP, and 2 mM beta-mercaptoethanol. This mixture was incubated end-over-end at 4 °C for 10 min. After centrifugation at 3,000 times g, supernatant containing Rab5 was collected. The DEAE-cellulose was washed 8 times, and the supernatants from all the washes were combined and concentrated at 4 °C, using micro-ProDiCon dialysis membrane (Spectrum). Purity was 95% as judged by Coomassie Blue staining. Aliquots were stored at -80 °C until use.

Intrinsic Tryptophan Fluorescence Measurements

Frozen aliquots of Rab5 proteins were quickly thawed and exchanged into buffer A (50 mM Hepes-HCl, pH 7.4, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% CHAPS) using a Bio-Gel P-6 spin column. Protein concentrations were estimated by the Bradford method(27) . In order to directly compare fluorescence intensities of GDP- and GTPS-bound forms of each protein, equivalent aliquots were removed from the same gel-filtered preparation to load with guanine nucleotide in parallel. Thus, any error in protein estimation is systematic within a given experiment comparing GDP- or GTPS-bound forms. Samples containing 500 nM protein, 20 µM GDP or GTPS (Boehringer Mannheim), and 0.5 mM MgCl(2) were incubated at 37 °C in buffer A for 2-3.5 h prior to fluorescence measurements. Preliminary experiments confirmed that 0.5 mM MgCl(2) promotes optimal binding for both guanine nucleotides and that complete exchange of nucleotide occurs under these conditions. Fluorescence emission spectra were recorded with a Hitachi model F-2000 spectrophotometer. The excitation wavelength was 290 nm, and emission data between 300 and 400 nm were collected at room temperature. The emission spectra of buffer A in the absence and presence of guanine nucleotide are negligible. The wavelengths of emission maxima were determined using software provided with the instrument; briefly, these values are derived from the spectrum based on ascending and descending slopes of a ``peak'' greater than 3 nm. Control experiments performed using an SLM Aminco Bowman Series 2 luminescence spectrometer and polarizer under ``magic angle conditions'' verified that the reported changes are not due to polarization artifacts.

NBS Oxidation Assay

As described above, samples from the same stock of protein were loaded in parallel with GDP or GTPS. NBS, freshly prepared in 50 mM Tris acetate, pH 5.0, was then added, and incubation was continued at room temperature in the dark for 10 min. The oxidation reactions were quenched by rapid addition of 2.5 µl of 1 M dithiothreitol. Fluorescence measurements were subsequently recorded as described above. The fluorescence intensity of each sample was converted to a percentage of the initial fluorescence (no NBS added) and plotted against the molar ratio of NBS to protein.

Miscellaneous

GTP-binding protein sequences were aligned using the GCG Package, version 7.3 (Genetics Computer Group, Madison, WI).


RESULTS

To examine the fluorescence properties of Rab5, samples of the protein were loaded in parallel with GDP and GTPS exactly as described under ``Experimental Procedures,'' and tryptophan fluorescence emission spectra were recorded. As shown in Fig. 2, the GTPS-bound form exhibits a blue shift relative to the GDP-bound form of the protein (maxima are at 336 and 339 nm, respectively). Moreover, the fluorescence intensity is markedly quenched in the GTPS-bound state. Results obtained in several identical experiments are summarized in Table 1, with the fluorescence intensity normalized to that measured for GDP-bound samples.


Figure 2: Emission spectra of wild type Rab5 in GTPS- and GDP-bound states. An aliquot of Rab5 was rapidly thawed and exchanged into buffer A, and protein concentration was determined as described under ``Experimental Procedures.'' Equivalent amounts of protein were removed from this stock to load with GDP or GTPS in parallel. Protein (500 nM) was incubated for 2 h at 37 °C in buffer A in the presence of 20 µM guanine nucleotide (GTPS or GDP) and 0.5 mM MgCl(2). Fluorescence emission spectra were recorded at an excitation wavelength of 290 nm. Fluorescence intensities are shown in arbitrary units and the (max) values for the GTPS- and the GDP-bound forms of the protein are 336 and 339 nm, respectively. The ratio of the peak intensity of the GTPS- and the GDP-bound forms is 0.74.





To identify whether the fluorescence properties of one or both of Rab5's tryptophan residues are affected by guanine nucleotide exchange, Rab5 and Rab5 were constructed by site-directed mutagenesis. As shown in Fig. 3and summarized in Table 1, the emission maxima for the two variant Rab5 molecules are also blue-shifted when they are bound with GTPS, indicating that environmental changes for both Trp and Trp must contribute to the blue shift observed for wild type. The fluorescence intensity is markedly quenched for Rab5, indicating that the local environment of Trp is altered upon guanine nucleotide exchange. The right panels of Fig. 3compare the differences in (max) between wild type and the two Trp Phe variants by normalizing their intensities to the same level in the GDP- and GTPS-bound states. The (max) of Rab5 is red-shifted while Rab5 is blue-shifted relative to Rab5 in both guanine nucleotide binding states (see also Table 1). This difference in the emission maxima between the two variants indicates that Trp may be located in a more non-polar environment than Trp.


Figure 3: Emission spectra of Rab5, Rab5, and Rab5. Rab5 proteins were rapidly thawed and exchanged into buffer A, and protein concentrations were determined as described under ``Experimental Procedures.'' For each protein, equivalent aliquots were were removed from this starting stock to load with GDP or GTPS in parallel. Proteins (500 nM) were incubated for 2 h at 37 °C in buffer A in the presence of 20 µM GDP or GTPS and 0.5 mM MgCl(2). Fluorescence emission spectra were then recorded as described for Fig. 1. Left panels show spectra for Rab5 (top) and Rab5 (bottom) in the GDP- and GTPS-bound states as indicated. Right panels present fluorescence spectra of Rab5 and Rab5 normalized to that of Rab5 in the GDP-bound (top) and GTPS-bound (bottom) forms. The (max) values for the GDP-bound forms are: Rab5, 339 nm; Rab5, 336 nm; Rab5, 342 nm; and for the GTPS-bound forms the (max) values are: Rab5, 336 nm; Rab5, 334 nm; Rab5, 340 nm.



It is important to note that our interpretation of the fluorescence data is based on the assumption that the Trp Phe conversions do not alter the Rab protein's structure. The two independent Trp Phe substitutions do not functionally impair guanine nucleotide binding capacity as demonstrated by overlay blots of [P]GTP binding (data not shown); however, we cannot exclude the possibility for minor conformational alterations that might contribute to the fluorescence properties of each variant. For example, data to suggest that Trp's environment is more hydrophobic relative to that of Trp could simply reflect subtle conformational differences in the altered protein(s). Nonetheless, our conclusion that both regions of Rab5 undergo conformational changes upon guanine nucleotide exchange is strongly supported by the spectral differences between the GDP- and GTPS-bound forms observed for both Rab5 and Rab5, as well as Rab5. A second caveat to the interpretation of our data involves the possibility of ligand-induced aggregation contributing to changes in the proteins' fluorescence properties. To examine potential artifacts caused by ligand binding, light scattering peak intensities were determined for each protein in the GDP- and GTPS-bound states. Because significant differences were not detected and no obvious trend was apparent with respect to GTPS binding (data not shown), we conclude that the formation of large protein aggregates does not occur. Although lower order ligand-induced oligomerization cannot be excluded, biochemical characterization of Rab5 (11) and other Rab proteins (28) has yet to reveal functional evidence to suggest this possibility.

To provide further support for our conclusions, the sensitivity of Rab5's tryptophans to NBS oxidation was measured. Since highly accessible residues typically are located on the protein surface or in a hydrophilic pocket, resistance to NBS oxidation indicates sequestration of a tryptophan within a more buried, hydrophobic environment. Fig. 4shows the dose-dependent loss of tryptophan fluorescence as a function of the molar ratio of NBS to protein. A measurable increase in resistance to oxidation was observed for the GTPS-bound form relative to the GDP-bound structure of each protein, consistent with the blue shift of the fluorescence emission maxima upon GTPS binding (Table 1). Although it is possible that modification of other amino acids by NBS at high concentrations might cause conformational effects to influence the intrinsic tryptophan fluorescence, we presume these alterations would have an equivalent contribution to measurements made for the GDP- and GTPS-bound forms of each protein. If this assumption is valid, then the NBS oxidation results indicate that upon GDP/GTPS exchange, both tryptophans are sequestered into more nonpolar surroundings.


Figure 4: NBS oxidation of Rab5, Rab5, and Rab5. Rab5 proteins were exchanged into buffer A; equivalent samples were removed and then loaded in parallel with 20 µM GDP (open symbols) or GTPS (filled symbols) as described for Fig. 3. NBS, freshly prepared in 50 mM Tris acetate, pH 5.0, was then added to samples at the indicated molar ratios, and oxidation reactions were carried out as described under ``Experimental Procedures.'' The fluorescence intensity of each sample was converted to a percentage of the initial fluorescence intensity (no NBS added) and plotted against the ratio of NBS/protein. circles, Rab5; squares, Rab5; triangles, Rab5.




DISCUSSION

Based on the results of our fluorescence experiments, we conclude that Rab5 undergoes conformational changes upon guanine nucleotide exchange in two regions of the molecule as reported by spectral differences noted for Trp and Trp. It is anticipated that the local environment of Trp should be altered upon GTPS binding due to its proximity to Rab5's cognate switch II domain. This prediction is based on the conformational changes noted in crystal structures of Ras(16) , EF-Tu(17) , and G(18) . However, the guanine nucleotide-dependent fluorescence properties of Trp are more informative. The sequence alignment of Fig. 1reveals that Trp is in a domain of Rab5 potentially related to the flexible switch III region of G. In fact, molecular modeling by Stenmark et al.(29) predicts nucleotide-dependent conformational changes in three structural elements of Rab5 upon GDP/GTP exchange, including L2 (the predicted switch I or effector loop), L4/alpha2/L5 (cognate to switch II), and alpha3/L7 (Fig. 1). The fluorescence characteristics we observe for Trp provide further experimental evidence demonstrating conformational changes in the alpha3/L7 region of Rab5 upon GTPS binding.

Switch III of G is a loop domain that detects molecular changes in switch II primarily via ionic interactions (18) . In the GTPS-bound state, switch II and III become juxtaposed to close a cavity in G's tertiary structure. While the alpha3/L7 domain of Rab5 lacks the charged residues highly conserved among G protein alpha-subunits, it may nonetheless functionally mimic the switch III domain to swing closer to L4/alpha2/L5 upon GTPS binding, thereby occluding a cleft in Rab5's tertiary structure. It is important to note that alpha3/L7 corresponds to domains in Ras and EF-Tu, which are also rearranged in response to molecular changes in the switch II domain of these GTP-binding proteins (16, 17) (for review, see (30) ). Sequence and structure alignment by Lambright et al.(18) place the latter domains immediately adjacent to the switch III loop of G.

Our prediction that alpha3/L7 fulfills a ``switch III-like'' function is based on the fluorescence properties of Rab5, which reveal environmental changes for both Trp and Trp upon GTPS binding. We envision that in the GTPS-bound state, L4/alpha2/L5 and alpha3/L7 may closely align with one another, mimicking structural changes observed for switch II and switch III domains of G(18) . This conformational change would create a hydrophobic pocket within which Rab5's tryptophans become buried. The latter idea is supported by the blue shift in (max) observed for both Trp and Trp in the GTPS-bound state. The fact that these residues become resistant to NBS oxidation upon GDP/GTPS exchange is a further indication that both tryptophans become sequestered.

Compelling functional similarities between heterotrimeric G protein alpha-subunits and Rab proteins support the structural rearrangement of ``switch II'' and ``switch III-like'' domains in Rab5. In the GDP-bound conformation, both switch II and III domains of G are solvent-exposed, lining a cavity that is thought to serve as a binding site for G(18) . This heterotrimeric complex associates with rhodopsin and, upon activation, the receptor catalyzes GDP/GTP exchange and the dissociation of G from G. Hence, the closure of the switch II and III domains is thought to result in dissociation of the G subunit from G. By analogy, we theorize the functional counterpart of G, guanine nucleotide dissociation inhibitor (GDI), may interact with Rab proteins in a similar fashion. It is well established that GDI binds to the prenylated GDP-bound forms of Rab proteins(31) . Bound GDI strongly inhibits GDP release(32, 33) and delivers the Rab proteins to the correct membrane compartment(14, 34, 35, 36) . GDI dissociates upon catalyzed GDP/GTP exchange of the Rabs at the membrane surface (14, 35) but will retrieve the GDP-bound proteins from the membrane after GTP hydrolysis(37, 38) .

Given the nature of the strong functional relationships between Rab/GDI and G/G, the ``switch II'' and ``switch III'' domains of GDP-bound Rab5, as identified by Trp and Trp, are likely to provide a binding pocket for GDI. This hypothesis is consistent with functional evidence indicating that both L4/alpha2/L5 and alpha3/L7 domains of Rab5 are required for membrane association(28) . Our model also is in agreement with the proposition that equivalent structural domains in GDP-bound Ras may interact with signaling molecules that become activated, much like G, only after GDP/GTP exchange(29) . Since among the Ras superfamily, only Rab, Rho, and Rac family members are known to interact with GDIs(31, 32, 39, 40, 41) , it is interesting to further speculate that all of these GTP-binding proteins may display structure-function relationships similar in nature to those of heterotrimeric G proteins.


FOOTNOTES

*
This work was supported by Grant CB-15 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Junior Faculty Award from the American Cancer Society. To whom correspondence should be addressed: Dept. of Nutrition, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. Fax: 617-432-2435.

(^1)
The abbreviations used are: GDI, guanine nucleotide dissociation inhibitor; EF, elongation factor; GTPS, guanosine 5`-O-(3-thiotriphosphate); NBS, N-bromosuccinimide; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; WT, wild type.


ACKNOWLEDGEMENTS

We thank the Molecular Biology Computer Research Resource of the Dana Farber Cancer Institute and in particular, Tom Graf, for assistance in sequence analysis and the laboratory of Dr. John Gollan at the Brigham and Women's Hospital, particularly Drs. Stephen Zucker and Michael Fuchs, for providing assistance with control fluorescence measurements made under magic angle conditions. We also appreciate the advice on these fluorescence measurements given by Dr. Ruth Sornoff at Hitachi Instruments and Dr. Joseph Lakowicz at the University of Maryland School of Medicine.


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