©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of Mg on the Structure and Function of Rab5 (*)

(Received for publication, August 29, 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
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mg inhibits GDP release from Rab5 but not from Rab5, a mutant lacking Ser critical for Mg coordination in the nucleotide binding pocket. Thus, inhibition of GDP release is apparently exerted via coordination of Mg between Rab5 and GDP. Mg also induces conformational changes in Rab5, demonstrated by increased tryptophan fluorescence intensity and a red shift in (max) for the GDP-bound protein. Mg-induced fluorescence changes are not observed for Rab5. The correlation between Mg effects on nucleotide exchange and the fluorescence properties of Rab5 suggests that a conformation promoted through Mg coordination with Ser also contributes to inhibition of GDP release. The role of structural changes in GDP release was investigated using C- and N-terminal truncation mutants. Similar to Rab5, Mg inhibits GDP release and alters the fluorescence of Rab5 but only partially inhibits release from Rab5 and fails to induce changes in the latter's fluorescence properties. Since Rab5 maintains Ser necessary for Mg coordination, the lack of Mg-induced fluorescence changes suggests a requirement for the N-terminal domain to promote a conformation blocking GDP release. A model for mechanisms of interaction between Ras-like proteins and their exchange factors is proposed.


INTRODUCTION

Rab proteins are a family of Ras-like small molecular weight GTPases that are localized to distinct subcellular compartments(1, 2) and believed to regulate specific steps of intracellular membrane trafficking(3, 4, 5, 6) . The functional cycle of Rab proteins involves the delivery of the GDP-bound forms to the target membrane by a GDP dissociation inhibitor (GDI)(^1)(7, 8, 9) , the exchange of GDP for GTP at membrane surface catalyzed by a guanine nucleotide exchange factor (GEF) (8, 9) and the retrieval of the GDP-bound forms from the membrane by GDI after GTP hydrolysis and membrane fusion(7) . Localized on plasma membrane, clathrin-coated vesicles, and early endosomes(2) , Rab5 has been shown to play an important role in early events of endocytosis(4, 5) , although the exact mechanism of its function remains to be determined.

It is known that Mg is essential for GTPase function and structure. Crystallographic studies of several GTP-binding proteins reveal a single Mg in the guanine nucleotide binding pocket, coordinating between the protein and guanine nucleotide in both GDP- and GTP analog-bound conformations(10, 11, 12, 13, 14, 15) . Effects of Mg on guanine nucleotide binding, GTPase activity, and the structural integrity of GTP-binding proteins have been widely documented(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) . A key observation is that Mg inhibits GDP release from Ras-like GTP-binding proteins and therefore prevents binding of GTPS(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . However, the exact mechanism for this inhibitory effect and its physiological significance remains unknown.

Important functional roles of the N-terminal domains of several Ras-like GTP-binding proteins also have been noted in studies of guanine nucleotide exchange(33, 34, 35, 36, 37, 38) . Myristoylation at the N terminus of ARF enhances its rate of GDP release(27) , and N-terminal truncation of ARF results in loss of function by reducing its affinity for GDP and permitting GDP/GTP exchange in the absence of phospholipids(33) . Moreover, deletion of the N terminus enables isolation of ARF in a nucleotide-free form(38) . Finally, deletion of the N-terminal domain of Rab5 results in a loss of function (34, 35, 36, 37) and interferes with the protein's post-translational processing(37) . These observations suggest that N-terminal domains of Ras-like GTP-binding proteins may participate in the regulation of guanine nucleotide exchange and represent crucial structural domains necessary for the function of the proteins.

We have investigated mechanisms through which Mg and the N-terminal domain of Rab5 participate in its regulation of GDP release. Physiologic concentrations of Mg block GDP release from Rab5 but not from Rab5, a mutant lacking Ser critical for Mg coordination. Mg also alters the intrinsic tryptophan fluorescence properties of Rab5 but not Rab5. While the structure and function of Rab5, a C-terminal truncation mutant, is influenced by Mg in the same fashion as Rab5, an N- and C-terminal truncation mutant, Rab5, is resistant to the cation's effects. Thus, inhibition of GDP release by Mg appears to be exerted via chemical constraints due to the cation's coordination between GDP and Rab5, as well as conformational restraints involving the protein's N-terminal domain that are induced by Mg coordination with Ser of Rab5. Based on the correlation between inhibition of GDP release and conformational changes promoted by Mg, we propose that in vivo, Mg prevents GDP dissociation from Rab5 until a guanine nucleotide exchange factor acts to promote GDP/GTP exchange, perhaps through interactions with the GTP-binding protein's N-terminal domain.


MATERIALS AND METHODS

Reagents

[S]GTPS, [^3H]GDP and [S]methionine were purchased from DuPont NEN. Unlabeled guanine nucleotides, CHAPS, trypsin, and restriction enzymes for subcloning were from Boehringer Mannheim. Rabbit reticulocyte lysate was from Promega.

Construction of Rab5 Mutants

Rab5 was amplified by polymerase chain reaction from wild-type Rab5 using the oligonucleotides 5`-CGCGGATCCATATGGCTAGTCGAGGC-3` and 5`-CCGTCGACTTATCCTCCTCCTCT-3`. The polymerase chain reaction product was subcloned into pAGA between BamHI and SalI sites. Similarly, Rab5 was amplified using oligonucleotides 5`-GGAATTCATATGCTAGTACTTCTGGGA-3` and 5`-CCGTCGACTTATCCTCCTCCTCT-3` as primers and subcloned into pAGA between EcoRI and SalI sites. The vectors containing Rab5 and Rab5 were digested with NdeI and SalI, and the excised fragments were also inserted into pT7.7 by directional subcloning. A point mutant Rab5 was constructed by the Kunkle method using oligonucleotide 5`-AAGCACTAGGCTGTTTTTGCCAACAGC-3` and also inserted into pT7.7. The construction of Rab5 in pAGA and Rab5 and Rab5 in pT7 were described previously(37, 39) . Sequences of all the Rab5 mutants were confirmed by the dideoxy chain determination method of Sanger.

In Vitro Transcription and Translation

Procedures for in vitro transcription and translation of nascent peptides in rabbit reticulocyte lysate have been described in detail(40) . Briefly, 10 µg of linearized pAGA containing wild-type or mutant cDNAs was transcribed using T7 polymerase. Translation of the transcripts was conducted in rabbit reticulocyte lysate in the presence of 10^6 cpm/µl [S]methionine (1200 Ci/mmol) at 30 °C for 20 min, and expressed proteins were quantified by trichloroacetic acid precipitation.

Proteolysis of in Vitro Synthesized Peptides

Proteolysis of in vitro translation products was as described by Sanford et al.(37) . Briefly, translated peptides were preincubated at 30 °C for 45 min in the absence and presence of 10 mM guanine nucleotide or 25 mM EDTA in 80 mM Tris-Cl, pH 8.0, 2 mM MgCl(2), 100 mM NaCl, and 1 mM dithiothreitol. Proteolysis was subsequently initiated by the addition of trypsin. The reaction was quenched with soybean trypsin inhibitor, and digested products were analyzed on a urea/acrylamide gradient SDS gel.

Expression and Purification of Recombinant Wild-type and Mutant Rab5

Procedures for protein expression and purification have also been described in detail previously(39) . Briefly, pT7.7 containing wild-type or mutant Rab5 cDNA was transformed into BL21(DE3) cells by electroporation. Cells were grown in TB in the presence of 50 µg/ml carbenicillin, and protein expression was induced by 0.8 mM isopropyl-1-thio-beta-D-galactopyranoside. Rab5 or mutant proteins present in the cell lysate were purified by ion-exchange chromatography using DEAE-cellulose (Whatman). Fractions containing Rab5 proteins were concentrated by vacuum dialysis, and the final protein concentration was determined by the method of Bradford. Samples were frozen at -80 °C until use.

Measurement of [^3H]GDP Dissociation Rates

Purified recombinant wild-type or mutant Rab5 protein was rapidly 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. Five µM Rab protein was incubated at 37 °C for 30 min with 200 µM [^3H]GDP (specific activity, 2.2 times 10^14 cpm/mol) in buffer A to preload with [^3H]GDP. Control samples containing the same amount of protein and [^3H]GDP but with 20 mM unlabeled GDP were incubated in parallel to determine nonspecific binding. After equilibration at room temperature, 15 µl of each reaction mixture was filtered to measure the initial amount of bound [^3H]GDP; excess unlabeled GDP (20 mM) and MgCl(2) (0-5 mM) were then added. At indicated time points, aliquots containing the same amount of protein were removed and filtered through nitrocellulose discs. Filters were washed 3 times with 2 ml of ice-cold wash buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM beta-mercaptoethonol) and radioactivity bound to protein trapped on each filter was measured by scintillation counting. Specific binding was determined as the difference in cpm measured in samples incubated with or without excess GDP.

Intrinsic Tryptophan Fluorescence Measurements

Proteins were thawed and exchanged into buffer A as described above. Wild-type or mutant Rab5 was incubated for 30 min at 37 °C with 100 µM GDP and increasing amounts of MgCl(2) in buffer A (total volume, 500 µl). Fluorescence emission spectra were recorded with an Hitachi model F-2000 spectrophotometer. Excitation wavelength was 290 nm and emission data between 300 and 400 nm were collected at room temperature.


RESULTS

Mg Inhibits GDP release from Rab5 but Not from Rab5

Mg has been shown to inhibit GDP release from many Ras-like GTP-binding proteins(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . Consistent with these observations, the results presented in Fig. 1A demonstrate that the rate of GDP dissociation from Rab5 is also dependent on [Mg]. In comparison with GDP dissociation in the absence of any added Mg, the addition of 5 mM Mg to the assay completely blocks the release of GDP, while little effect is observed with 0.5 mM Mg. The values of k determined from the slopes of these lines are presented in Table 1. The inhibitory effect is reflected in the markedly reduced k at [Mg] > 1.5 mM (data not shown). Therefore, under these assay conditions (Table 2), we estimate that micro- to millimolar levels of free Mg strongly inhibit GDP release, whereas free Mg in the submicromolar range does not.


Figure 1: Effects of Mg on the rate of [^3H]GDP dissociation from Rab5 and Rab5. Purified Rab5 (panel A) and Rab5 (panel B) (5 µM) were incubated at 37 °C for 30 min with 200 µM [^3H]GDP (specific activity, 2.2 times 10^14 cpm/mol). Control reactions containing the same amount of protein and [^3H]GDP, but with 20 mM unlabeled GDP were incubated in parallel to measure nonspecific binding. After equilibration at room temperature, an aliquot of each sample was filtered to determine the amount of [^3H]GDP bound. Excess unlabeled GDP (20 mM) with 0.5 mM (triangle) or 5 mM (circle) or without (square) Mg were then added, and GDP dissociation was monitored at room temperature. An aliquot of each sample was filtered at indicated time points, and the amount of [^3H]GDP bound was determined by scintillation counting. Specific binding was determined as difference in cpm measured in samples incubated with or without excess GDP. Shown is the mean of duplicate measurements plotted as a function of ln (B/B(0)) against time (B(0), initial specific binding; B, specific binding at each time point). Identical results were obtained on three separate occasions.







To investigate the mechanism through which Mg inhibits GDP release, the point mutant Rab5 was constructed by site-directed mutagenesis. Ser of Rab5 is expected to coordinate with Mg in the guanine nucleotide binding pocket in both GDP- and GTP-bound states based on crystallographic evidence obtained for other GTP-binding proteins(10, 11, 12, 13, 14, 15) . The cognate mutant of Ras at this position, Ras, displays an accelerated rate of GDP release that is insensitive to Mg(20) . Fig. 1B demonstrates that Rab5 also exhibits a rate of GDP dissociation greater than Rab5, but more importantly, the release of GDP is no longer affected by Mg. As summarized in Table 1, this effect is reflected in the large difference in k determined in the presence of high Mg (0.245 versus 0.006 min for Rab5). Thus, it is likely that coordination of Mg with Ser participates in a direct chemical restraint preventing dissociation of the nucleotide. However, it is also possible that Mg could promote structural rearrangements in Rab5 through interactions with Ser, resulting in a protein conformation that is unfavorable for GDP release. These two mechanisms are not mutually exclusive, and both may contribute to the observed inhibitory effect of Mg.

Mg Alters the Intrinsic Tryptophan Fluorescence of Rab5, but Not of Rab5

To examine whether protein structural changes are induced by Mg, we investigated its effects on the intrinsic tryptophan fluorescence of Rab5; the latter provides a sensitive probe for local conformational changes in GTP-binding proteins(41, 42, 43, 44, 45) . We previously characterized guanine nucleotide-dependent fluorescence changes for Rab5 (39) . As shown in Fig. 2, Mg causes a dramatic increase in the tryptophan fluorescence intensity of GDP-bound Rab5. The increase in fluorescence intensity is accompanied by a slight red shift in (max). Further examination of Mg effects on the individual fluorescence properties of Rab5 and Rab5 indicates that each of the two tryptophans of Rab5 contributes to the observed fluorescence changes in Rab5 (data not shown). Our results suggest that Mg induces conformational changes in GDP-bound Rab5 that place both of its tryptophans into a more polar environment. Since the inhibitory effect on GDP release and the induced structural changes in Rab5 occur within the same range of [Mg], we postulate the increased fluorescence is due to an altered form of the protein that is unfavorable for GDP release. To test this hypothesis, we examined the influence of Mg on the fluorescence properties of Rab5. In contrast to Rab5, neither the fluorescence intensity (Fig. 3) nor (max) of Rab5 (data not shown) is affected by high [Mg]. This result supports the idea that protein conformation promoted by the coordination between Mg and Ser plays an important role in the inhibition of GDP release.


Figure 2: Effects of Mg on the tryptophan fluorescence properties of Rab5. Purified Rab5 (400 nM) was incubated in buffer A at 37 °C for 30 min with 20 µM GDP and indicated amounts of MgCl(2). Fluorescence emission spectra were recorded between 300 and 400 nm for each sample at an excitation wavelength of 290 nm. Shown is a representative results from at least three separate experiments. The (max) values determined for Rab5 in the absence and presence of 5 mM Mg were, respectively, 340-341 and 342-343 nm.




Figure 3: Effects of Mg on the fluorescence intensities of Rab5 and Rab5. Purified Rab5 (filled squares) and Rab5 (open squares) (0.5 µM) were incubated in buffer A at 37 °C for 30 min with 100 µM GDP and increasing amounts of MgCl(2). Fluorescence emission peak intensity and wavelength was recorded for each sample at an excitation wavelength of 290 nm. The fluorescence intensity of each sample was normalized against the measurement of protein without Mg and plotted as a function of added Mg concentration. Shown is the mean of duplicate measurements. Similar results were obtained on at least three separate occasions.



Characterization of Rab5 Mutants by Protease Protection Assays

Several lines of evidence suggest potential roles for the N- and the C-terminal domains of small GTP-binding proteins in Mg-induced conformational changes and the regulation of guanine nucleotide exchange. Ser is located within the N terminus, thus it is easy to envision that coordination between Mg and Ser could promote conformational rearrangements within this domain. Myristoylation at the N terminus and truncation of the N-terminal domain of ARF alter the protein's affinity for GDP and properties of guanine nucleotide exchange(33, 38) . In addition, N-terminal truncation interferes with the function of both Rab5 and ARF (33, 34, 35, 36) as well as post-translational processing of Rab5(37) . Finally, post-translational modification of Ras-like proteins at their C-terminal domains, including phosphorylation, isoprenylation, and carboxyl methylation, is important for GDP dissociation stimulator action(46, 47) .

To evaluate the relative roles of the N and the C termini as potential regulators of guanine nucleotide exchange, three truncation mutants of Rab5 were characterized (Rab5, Rab5, and Rab5). Filter binding assays with [^3H]GDP and [S]GTPS indicated that both Rab5 and Rab5 bind guanine nucleotides in solution, although Rab5 exhibits a reduced affinity for both nucleotides (data not shown). Previous studies demonstrated that Rab5 binds [^3H]GDP(38) . Further information on the tertiary structure of these proteins was obtained from protease protection assays(37) . As shown in Fig. 4, in vitro synthesized S-labeled Rab5 migrates as a 27 kDa band on a urea/acrylamide gradient SDS gel. The synthetic protein binds endogenous GDP(40) ; limited digestion of this GDP-bound form with trypsin produces a single S-labeled fragment of 14 kDa. Addition of 30 mM EDTA to chelate Mg essential for guanine nucleotide binding markedly reduces the amount of the latter tryptic peptide. However, incubation with GTPS prior to trypsinization results in protection of a S-labeled 20-kDa peptide in addition to the 14-kDa fragment, representing a ``core'' structure of Rab5 in the GTPS-bound conformation. Fig. 4further demonstrates that protease protection profiles for Rab5, Rab5, and Rab5 are all similar to Rab5, confirming that each of the truncation mutants binds guanine nucleotides.


Figure 4: Protease protection of N- and C-terminal deletion mutants of Rab5 by guanine nucleotides. In vitro synthesized S-labeled Rab5, Rab5, Rab5, and Rab5 were incubated at a final concentration of 3.5 nM for 45 min at 30 °C with or without 10 mM GTPS or 30 mM EDTA as indicated. Proteolysis was initiated by addition of 0.022 units of trypsin, and incubations were continued for 45 min. Digestion was terminated with 2 µg of soybean trypsin inhibitor at 20 °C for 5 min. Samples were diluted with 68 µl of Laemmli buffer, heated for 5 min at 100 °C, and electrophoresed on urea/acrylamide gradient SDS gels. Shown is fluorography of the dried gel (2-day exposure); tic marks indicate the migration of 20- and 14-kDa markers. Mass of S-labeled tryptic fragments was determined as follows: Rab5, 14 and 20 kDa; Rab5, 14 and 20 kDa; Rab5, 14 and 18.3 kDa; and Rab5, 14 kDa and 18.3 kDa. The lowest molecular weight species detected for Rab5 and Rab5 is not a tryptic peptide fragment since it appears in the(-) trypsin lanes.



By comparing the sizes of the GDP- and GTPS-protected fragments of Rab5 and these truncation mutants, a tryptic cleavage map can be predicted as shown in Fig. 5. Our assignments are made on the following premises. Since both of the GTPS-protected fragments for Rab5 and Rab5 are identical and approximately equal in size to undigested Rab5, a C-terminal proteolysis site must exist upstream of, but close to, position 198. The only practical assignment is Arg. Trypsinization of the GDP-bound forms of all the Rab5 molecules produces identical 14-kDa fragments, indicating that cleavage sites must be downstream of position 23 and upstream of position 198. The fact that this peptide is heavily radiolabeled suggests that several methionine residues are present. Based on the size of this tryptic fragment, Lys and Arg are the most likely tryptic sites since cleavage at these residues would generate a 13.8-kDa fragment. Since the GTPS-protected fragments of Rab5 and Rab5 migrate with a mass 1.7 kDa larger than that of Rab5 on SDS-gels, if there is an N-terminal tryptic site, it would be best positioned at Arg^4 or Arg^8. However, because of the proximity of these residues to the N terminus, and given the vagaries of peptide mobilities on SDS gels, we cannot distinguish which Arg provides the tryptic cleavage site nor can we confirm that proteolysis at the extreme N terminus occurs. It should be noted that Steele-Mortimer and co-workers (35) have previously assigned Arg^4 as a tryptic site for Rab5 by N-terminal sequencing (35) . A key prediction from the proposed tryptic map is that GTPS-binding protects against proteolytic cleavage at Lys, supporting the idea that the Rab5's N-terminal domain undergoes conformational rearrangement upon GDP/GTP exchange.


Figure 5: Schematic diagram of predicted sites of trypsin proteolysis in the GDP- and GTPS-bound forms of Rab5. Shown is a linear model of the Rab5 peptide backbone interrupted by solid bars depicting the putative guanine nucleotide binding domains. Locations of all potential tryptic sites are indicated by short vertical bars. Locations of methionine residues in the mature peptide are indicated by asterisks. Our proposed assignment of cleavage sites (long vertical bars) in the GDP- and GTPS-bound forms of Rab5 was based on determination of the mass of S-labeled tryptic peptides as shown in Fig. 4and discussed in the text. Lys and Arg are chosen as tryptic sites since cleavage would be predicted to generate a 13.8-kDa fragment and Arg is the most logical assignment as a C-terminal proteolysis site in all of the GTPS-bound forms. Alternative assignments that cannot be completely ruled out are at N-terminal residues Arg/Lys and C-terminal residues Lys/Lys/Lys; however, proteolytic cleavage at these sites would yield 15-kDa peptides. It should be noted that this alternative assignment does not argue against conformational changes protecting the N-terminal domain, it simply indicates that the Arg/Lys is the location of the protected cleavage site and that C-terminal conformational changes also might occur upon guanine nucleotide exchange.



In comparison with Rab5, Rab5 produces a distinct protease protection pattern (Fig. 6). In the absence of exogenously added guanine nucleotide, Rab5 is completely degraded by trypsin, unlike wild-type, which displays a 14-kDa fragment protected by endogenous levels of GDP. The accelerated rate of GDP release from the mutant (Fig. 1B) is most likely responsible for this effect, making Rab5 more susceptible to proteolysis. When saturating levels of GDP or GTP analogs are added, a 14-kDa fragment of Rab5 is protected. The absence of the 20-kDa core peptide in the GTP analog-bound forms suggests that Rab5 resembles the GDP-bound conformation of wild-type, consistent with previous reports characterizing the cognate mutants of other Rab proteins(48, 49, 50) . The failure of GTPS or Gpp(NH)p to protect the predicted Lys cleavage site also suggests that Mg coordination with Ser is necessary to induce conformational changes upon guanine nucleotide exchange.


Figure 6: Protease protection of Rab5 and Rab5. In vitro synthesized S-labeled Rab5 and Rab5 were incubated at a final concentration of 3.5 nM for 45 min at 30 °C with or without 10 mM guanine nucleotide analog (GTPS, Gpp(NH)p, or GDPbetaS) or 30 mM EDTA as indicated. Proteolysis was carried out exactly as described for Fig. 4; S-labeled tryptic fragments were 14 and 20 kDa for Rab5 and 14 kDa for Rab5.



Characterization of Mg Effects on Rab5 and Rab5

To further evaluate the structural role of the N-terminal domain of Rab5, the effects of Mg on GDP dissociation from Rab5 and Rab5 were compared. The data presented in Fig. 7A and summarized in Table 1show that GDP release from Rab5 is markedly inhibited by 5 mM Mg, similar to the results obtained for Rab. Thus, it is unlikely that the C-terminal domain of Rab5 participates in Mg-induced inhibition of GDP dissociation. In contrast, the ability of Mg to inhibit GDP release is partially lost when the N-terminal domain of Rab5 is truncated (Fig. 7B). Because all six coordinating ligands for Mg remain unperturbed in Rab5, chemical constraint(s) due to its chelation within the guanine nucleotide binding pocket most likely account for the residual inhibitory action of the cation. The reduced inhibition of GDP release suggests, however, that the ability of Rab5 to undergo structural changes in response to Mg-Ser coordination could be impaired due to the absence of the N-terminal domain.


Figure 7: Effects of Mg on the rate of [^3H]GDP dissociation from Rab5 and Rab5. [^3H]GDP dissociation of Rab5 (panel A) and Rab5 (panel B) were measured in the absence (square) and presence of 0.5 mM (triangle) or 5 mM (circle) added MgCl(2) as described in Fig. 1. Each data point is the mean of duplicate measurements. Identical results were obtained on three separate occasions.



To test this possibility, the influence of Mg on the fluorescence properties of Rab5 and Rab5 was also compared (Fig. 8). Like Rab5 the intrinsic fluorescence intensity of Rab5 is increased by Mg. In contrast to Rab5 but similar to Rab5, the fluorescence properties of Rab5 do not change in response to increasing [Mg]. Thus, in addition to chemical constraints exerted by Mg coordination within the guanine nucleotide binding pocket, the Mg-Ser link imposes structural restraints that involve the N-terminal domain and contribute to inhibition of GDP release.


Figure 8: Effects of Mg on the fluorescence intensities of Rab5 and Rab5 . Effects of Mg on the fluorescence properties of Rab5 (filled square) and Rab5 (open square) were measured as described in Fig. 2. Normalized fluorescence intensities were plotted against added Mg concentration as shown in Fig. 1. Similar results were obtained on at least three separate occasions.




DISCUSSION

Studies on crystallized Ras(10) , EF-Tu(11) , G(12) , ARF(13) , and Ran (14) in their GDP-bound forms have revealed identical Mg coordination within the guanine nucleotide binding pocket. The cation interacts directly with the beta-phosphate of GDP and a highly conserved Ser/Thr near the N terminus of these proteins; indirect interactions are mediated through four associated water molecules. In the GTP analog-bound forms of Ras(10) , EF-Tu(1) , G(12) , and G(15) , Mg coordination with two of these water molecules is replaced by a direct coordination with the -phosphate of the nucleotide and another highly conserved Thr. Inhibitory effects of Mg on GDP release have been documented for a wide variety of small GTP-binding proteins(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) , and our results extend this general observation to include Rab5. Mutation of Ser in Rab5 results in an increased rate of GDP release, which is no longer affected by Mg, a phenomenon also observed for the cognate mutants of other small GTP-binding proteins, including Ras(20) , Rab1(48) , Rab9 (49) , and Rab3(50) .

In vivo, the release of GDP from Rab5, and most likely other small GTP-binding proteins, would be inhibited by intracellular free Mg. Thus, a common mechanism to regulate guanine nucleotide exchange may be adopted by this family of proteins. In order to facilitate GDP/GTP exchange, a GEF must overcome the inhibitory restraints imposed by intracellular Mg levels. Through transient disruption of the Mg-Ser/Thr coordination, GEF could be envisioned to induce structural changes such that the GTP-binding protein adopts a conformation similar to its Ser Asn cognate mutant, thereby promoting GDP dissociation.

Several lines of evidence support this idea. Our fluorescence studies demonstrate that Rab5 adopts a Mg-insensitive conformation that is distinct from the Mg-sensitive conformation of Rab5. Although the strong dominant inhibitory effects of cognate mutants are generally attributed to their preferential affinity for GDP over GTP(6, 48, 49, 52, 53, 54, 55, 56, 57, 58, 59, 60) , Wittinghofer and co-workers (20, 63) have argued that the distinct structures of the Ser Asn cognate mutants are most likely responsible for disruption of wild-type protein function through competition for GEF interactions. The fact that Ras has an even greater affinity for GDP than Ras but is not a suppressor of Ras function supports this idea(20) . Moreover, Rab3A(50) , Ras(51) , and Ran(52) have been shown to have a much higher affinity for their respective GEFs than wild-type, further suggesting that complexes formed between the Ser Asn cognate mutants and their corresponding GEFs are thermodynamically more favorable. It has been proposed that the yeast GEF Cdc25p promotes exchange by stabilizing Ras in a nucleotide-free state(53) ; thus, it seems likely that Ser Asn cognate mutants of Ras-like factors are analogous to the nucleotide-free conformations of these proteins. The fact that exogenously added GDP and GTP analogs produce the same protease protection profile indicates that Rab5 must accommodate both nucleotides in its binding pocket but fails to undergo molecular rearrangements associated with guanine nucleotide exchange. This evidence supports the view that the mutant adopts a structure intermediate between GDP- and GTP-bound states. Ser is therefore predicted to be a critical residue involved in the conformational switch during guanine nucleotide exchange.

Our study demonstrates that Mg-Ser coordination between GDP and Rab5 not only provides a chemical constraint, but also promotes conformational changes involving the N terminus, which impose additional structural restraints against GDP dissociation. The markedly reduced inhibition of GDP release by Mg and the lack of Mg-induced fluorescence changes in Rab5 indicate that the N-terminal domain of Rab5 must play a key role in maintaining a conformation that blocks GDP release. It should be noted that the first 23 amino acids of Rab5 correspond to a rather small domain comprising only 4 residues in Ras based on pattern-induced multiple alignment analysis(37) . As previously noted in a comparison with Ras(36) , the N-terminal domain of Rab5 is one of five regions of this molecule predicted to impart functional specificity. Thus, although Ras(20) behaves in a fashion similar to Rab5, whether Mg-induced N-terminal conformational changes contribute to inhibition of GDP release from Ras is uncertain. Comparison of Ras crystal structures in the GDP and GTP analog-bound states reveals no obvious conformational changes in its N-terminal domain(10) , but this does not preclude the possibility of structural rearrangement during the guanine nucleotide exchange process. Since the N terminus of ARF also has been reported to participate in guanine nucleotide exchange regulation, it is possible that structural features of this domain may be shared among other Ras-like GTP-binding proteins. We speculate that GEF directly interacts with the N-terminal domain of Rab5 to interrupt Mg coordination with Ser, thereby promoting a transient conformation that facilitates GDP release. In support of this idea, truncation of the N-terminal domain abolishes the function of Rab5(34, 35) .

Since our data show that cellular levels of Mg are sufficient to block GDP release, what is the role of the guanine nucleotide dissociation inhibitor GDI? Even though this factor was purified based on its ability to inhibit GDP dissociation(64, 65) , under physiologic Mg concentrations its relative activity is only marginal(65, 66, 67) . Except for Rac (68, 69) and Rho(70) , similar factors have yet to be identified for other Ras family members. This suggests that a GDI per se is not required to prevent GDP dissociation from small GTP-binding proteins since cellular Mg levels are sufficient to provide this function. In fact, upon delivery of Rab proteins to membranes, exchange for GTP is not immediate after GDI dissociation(8, 9) . Our study indicates that during this time period, Mg most likely prevents GDP dissociation. Thus, we speculate that the true function of the Rab GDI may be one of a molecular escort protein or chaperone. In this capacity, GDI may stabilize the RabbulletMgbulletGDP complex, blocking interaction sites for GEF until the appropriate membrane target is reached. This function is comparable with that of the beta subunit complex of heterotrimeric G proteins, which also retards GDP release and targets alpha subunits to their appropriate membrane receptors. Indeed, our recent studies revealed structure-function similarities between Rab5 and the alpha subunits of GTP-binding proteins (39) . However, GDP release from most G subunits is unaffected by Mg. In fact, the G subunits contain a unique alpha-helical domain absent from Ras-like GTP-binding proteins that may block GDP release(12, 13) . The N-terminal domain of Rab5 may substitute, in part, for this structural element.


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; GEF, guanine nucleotide exchange factor; ARF, ADP ribosylation factor; GTPS, guanosine 5`-O-(thio)triphosphate; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; GDPbetaS, guanyl-5`-yl thiophosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonic acid; WT, wild type.


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