©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interferon-induced MxA Protein
GTP BINDING AND GTP HYDROLYSIS PROPERTIES (*)

Marc F. Richter , Martin Schwemmle , Christian Herrmann (1), Alfred Wittinghofer (1), Peter Staeheli (§)

From the (1) Abteilung Virologie, Institut für medizinische Mikrobiologie und Hygiene, Universität Freiburg, 79008 Freiburg, Germany Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, 44139 Dortmund, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min. The K for this reaction was 260 µM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (K 20 µM, k 8.5 s) than the nonhydrolyzable GTP analog mant-5`-guanylyl-,-imidotriphosphate (mant-GMP-PNP) (K 0.75 µM, k 0.012 s). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the K for GTP was 20 µM, whereas the K for GDP was 100 µM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.


INTRODUCTION

GTPases play key roles in fundamental cellular processes such as protein synthesis, intracellular signaling, and intra-cellular vesicle transport (1, 2, 3) . A common feature of most GTPases is that they can function as molecular switches; GTP- and GDP-bound forms have different conformations that influence their interactions with target proteins (4, 5) . The best studied GTPases are p21 and ras-related low molecular weight GTPases (6, 7, 8) which exhibit characteristically high binding affinities for GTP and GDP, have low intrinsic GTPase activity, and require accessory proteins for efficient hydrolysis of GTP and release of GDP. Among the high molecular weight GTPases, members of the G family (9) and subunits of the signal recognition particle receptor (10) show biochemical characteristics that are distinct from those of ras-related proteins. G proteins have a built-in GTPase-activating protein domain (11) which facilitates GTP hydrolysis and accounts for their high intrinsic GTPase activity. The signal recognition particle receptor has low affinity for guanine nucleotides, and it was postulated that under physiological conditions a significant fraction of signal recognition particle receptor molecules might exist in the empty state (10) . The biochemical properties of a newly emerging family of GTPases have been less well characterized to date. Besides Mx proteins, this family includes the dynamins of mammals and flies (12, 13, 14, 15) and Vps-1p of yeast (16, 17, 18) which are involved in regulating intracellular vesicle transport.

Mx proteins are synthesized by many cell types in response to virus-induced interferon (19) . Human MxA possesses intrinsic antiviral activity; expression of MxA cDNA in susceptible cells confers a high degree of resistance to certain RNA viruses, including influenza A, measles, vesicular stomatitis, and Thogoto virus (20, 21, 22, 23) . Transgenic mice expressing MxA show enhanced virus resistance,() indicating that MxA serves as an intracellular mediator of the interferon-induced antiviral state. The role of the MxA-associated GTPase activity in virus defense remains elusive. Mutational analysis showed that a functional GTP binding domain is required (25) . Since MxA inhibits virus transcription in vitro in the presence of GTP analogs that cannot be hydrolyzed by MxA (26, 27) , it seems that the binding of GTP rather than its hydrolysis is of critical importance. Purified MxA readily converts radiolabeled GTP to GDP; active MxA was purified successfully from interferon-treated human fibroblasts (28) , from insect cells infected with a recombinant baculovirus (29) , and from Escherichia coli transformed with a recombinant plasmid vector (25, 26) . GTP binding of MxA could be demonstrated by UV cross-linking experiments (25) , but further analysis of this activity was difficult. Filter binding assays suggested that MxA binds GDP with higher affinity than GTP (29) . However, this finding was difficult to reconcile with results from GTPase activity studies (25, 28, 29) .

To characterize the biochemical properties of MxA in more detail, we determined the parameters for GTP binding and GTP hydrolysis of purified recombinant histidine-tagged MxA (His-MxA).() Key features of His-MxA are its low affinity for GTP, its still lower affinity for GDP, and its high K for the GTP hydrolysis reaction.


EXPERIMENTAL PROCEDURES

Purification of His-MxA Protein from E. coli

His-MxA was purified from E. coli as described (25, 26) . Briefly, bacteria were transformed with an expression construct that codes for full-length MxA with additional amino acids at the NH terminus including six histidine residues. Purification of His-MxA to near homogeneity was achieved by affinity chromatography on nickel chelate agarose followed by Mono Q ion exchange chromatography.

Gel Filtration Chromatography

Purified His-MxA (1 mg/ml) was applied in a final volume of 500 µl to a Hi Load 16/6 Superdex 200 preparation grade gel filtration column (Pharmacia, Freiburg, Germany), equilibrated in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl, 10% glycerol, and 1 mM 2-mercaptoethanol). Protein was eluted with a flow rate of 0.5 ml/min, and fractions of 1.4 ml were collected. The elution profile of His-MxA was analyzed on 10% SDS-polyacrylamide gels by loading 30 µl of every second column fraction. To calibrate the column, 500-µl samples of bovine serum albumin (67 kDa), aldolase (158 kDa), and catalase (232 kDa) at 1 mg/ml were applied. The void volume was determined in a separate run by applying blue dextran 2000 (2,000 kDa). Samples of each second column fraction were tested for GTPase activity in a final volume of 50 µl of GTPase buffer consisting of 100 µM GTP, 13 nM [-P]GTP (3,000 Ci/mmol), 50 mM Tris-HCl, pH 8.0, 5 mM MgCl, 100 mM KCl, 10% glycerol, 0.1 mM dithiothreitol, and 100 nM AMP-PNP.

HPLC Analysis

Analysis of nucleotides by HPLC was performed as described (30, 31) with slight modifications. Briefly, a C-18 reversed phase column (0.4 x 25 cm filled with 5 µm ODS Hypersil, Bischoff, Leonberg, Germany) was run at ambient temperature with a flow rate of 1 ml/min in 50 mM sodium phosphate, pH 6.5, containing 0.2 mM tertiary butylammoniumbromide, 3% (v/v) acetonitrile, and 0.2 mM NaN. In this system, GDP eluted at 6.2 min and GTP at 8.2 min. To determine whether His-MxA was purified in nucleotide-bound form, a 10-µl sample of a 226 µM solution of His-MxA was applied to the HPLC column. Denatured protein was trapped on a precolumn (10 x 4.6 mm) filled with 5 µm ODS Hypersil. A 10-µl sample of a premixed solution that contained 20 µM GDP and 20 µM GTP was applied in a separate run as a control. The absorption was measured at 252 nm with a VWM-2141 UV detector (Pharmacia), and the signals were quantified with a C-R5A integrator (Shimadzu, Kyoto, Japan).

Nucleotides

The radiolabeled nucleotides [-P]GTP (3,000 Ci/mmol) and [-P]ATP (3,000 Ci/mmol) were purchased from Amersham, Braunschweig, Germany. The nonhydrolyzable nucleotide triphosphate analogs AMP-PNP, GMP-PNP, and GTPS were purchased from Boehringer Mannheim, Germany. GTP, ATP, UTP, and CTP were from Pharmacia. Fluorescent N-methylanthraniloyl (mant)-GDP and mant-GMP-PNP were synthesized and purified as described (32) with slight modifications (33) .

Assay for GTPase Activity of His-MxA

GTPase assays were performed with 0.1 mg/ml purified His-MxA in buffer B (13 nM [-P]GTP (3,000 Ci/mmol), 50 mM Tris-HCl, pH 8.0, 5 mM MgCl, 100 mM KCl, 10% glycerol, 0.1 mM dithiothreitol, and 100 nM AMP-PNP at 37 °C as described (26) . The concentration of unlabeled GTP was 1 mM, except where the indicated concentrations were used. At various times, the reaction was stopped by adding an equal volume of a stop solution containing 2 mM EDTA and 0.5% SDS. Samples were spotted onto polyethyleneimine-cellulose thin layer chromatography plates (MN300, Macherey und Nagel, Düren, Germany) and resolved in buffer C (1 M acetic acid, 1 M LiCl). The plates were exposed to x-ray film. The signals were quantified with a digital autoradiograph LB286 (Berthold, Wildbad, Germany). To calculate specific GTPase activities, time points within in the linear range of the hydrolysis reaction were used.

To estimate the pH optimum, the Tris buffer was adjusted to various pH values. To determine the temperature optimum, purified His-MxA was incubated for 10 min at 37 °C before nucleotides were added, and the mixture was incubated at various temperatures from 4 to 42 °C. The temperature stability was determined by incubating His-MxA for 20 min at various temperatures from 4 to 60 °C before nucleotides were added, and the mixture was incubated at 37 °C. To determine the K and turnover number, GTPase reactions were performed under optimized conditions. The GTP concentration varied from 0.05 to 1.75 mM.

Assay for ATPase Activity of His-MxA

ATPase assays were done like GTPase assays, except that ATP was used instead of GTP and that the buffer contained no AMP-PNP.

Nucleotide Binding Studies

Mant derivatives of GDP and GMP-PNP were prepared as described (32) with modifications as suggested (33) . Static and slow time scale dynamic fluorescent measurement were done on a Perkin-Elmer LS 50 B spectrophotometer (Perkin-Elmer, Buckinghamshire, U. K.). Excitation of mant nucleotides was at 366 nm, and emission was measured at 450 nm. For fast time scale experiments a stopped-flow apparatus (Hi-Tech Scientific, Salisbury, U. K.) was used. Here, detection of the emitted fluorescence was through a filter with a cutoff at 389 nm. Data were collected with an ADS analog-digital converter (Hi-Tech Scientific) and analyzed with the Hi-Tech Scientific software package on a personal computer. Secondary analysis of the static and dynamic data was done with the program GraFit (Erithakus Software) on a personal computer. All reactions were done at 25 °C in buffer D (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl, 10% glycerol, and 1 mM dithiothreitol).


RESULTS

Oligomerization State of His-MxA after Purification from E. coli

We described previously a simple method for the production of highly purified recombinant MxA protein (25, 26) . MxA cDNA was cloned into the bacterial expression vector pQE9 so that the recombinant protein carried six extra histidine residues at the NH terminus, which allows a single-step affinity purification with nickel chelate agarose. After chromatography on Mono Q, His-MxA was more than 98% pure. When applied to a S-200 gel filtration column, the bulk of His-MxA was found in the void volume and in early fractions (Fig. 1A). Very little His-MxA was found in fractions expected to contain the monomeric 76-kDa form of His-MxA. Additional experiments with a Superose 6 HR column (Pharmacia) indicated that the average size of the oligomers was about 2,000 kDa (data not shown), suggesting that they consisted of about 30 His-MxA monomers. The calculated specific GTPase activities of His-MxA in the various column fractions were indistinguishable (data not shown), indicating that the degree of oligomerization has no major effect on the GTPase activity of MxA. Other gel filtration experiments were performed in the presence of 1 M sodium chloride, in buffer containing 100 µM GMP-PNP or in buffer lacking detergents. The oligomerization status of His-MxA did not change under these conditions (data not shown). Thus, MxA produced in both E. coli and baculovirus-infected insect cells (29) formed high molecular weight oligomers.


Figure 1: His-MxA purified from E. coli forms oligomers and hydrolyzes GTP but not ATP. Panel A, oligomer formation. A sample (500 µg) of Mono Q-purified His-MxA was subjected to a S-200 gel filtration column. Fractions of 1.4 ml were collected, and samples of every second fraction were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. The migrations of bovine serum albumin (67 kDa), aldolase (158 kDa), and catalase (232 kDa) during calibration runs are indicated. Proteins larger than about 600 kDa eluted in the void volume. Panel B, GTPase activity. Samples of Mono Q-purified His-MxA (0.2 µg/µl) were incubated for 0, 17, or 34 min at 37 °C with either 1 mM unlabeled GTP and 13 nM [-P]GTP (3,000 Ci/mmol) or 1 mM unlabeled ATP and 13 nM [-P]ATP (3,000 Ci/mmol). The reaction products were analyzed by polyethyleneimine-cellulose thin layer chromatography and autoradiography. The positions of GTP, GDP, ATP, and ADP are indicated. Panel C, kinetic parameters of His-MxA GTPase. A substrate saturation experiment was carried out by incubating His-MxA (0.27 µg/µl) at 37 °C with increasing concentrations of GTP (0.05-1.75 mM). Samples were removed at various times and analyzed by polyethyleneimine-cellulose thin layer chromatography. The signals were quantitated using a digital autoradiograph, and the conversion rate of GTP to GDP was calculated. The Vversus [S] plot is shown. The calculated K was 260 µM. The GTP turnover number of our most active preparations was 27 min.



Enzymatic Parameters of the His-MxA-associated GTPase

Radiolabeled GTP incubated with samples of purified His-MxA was rapidly converted to GDP (Fig. 1B). ATP was not hydrolyzed (Fig. 1B), demonstrating that His-MxA exhibited a high degree of substrate specificity. Furthermore, this result showed that our preparations of His-MxA were essentially free of contaminating phosphatases. In a standard GTPase assay, hydrolysis of GTP increased linearly for at least 15 min and was directly proportional to the concentration of His-MxA. The hydrolysis reaction was dependent on magnesium, was rather insensitive for changes in pH from 6.9 to 8.3, and had a temperature optimum at about 37 °C. Incubating His-MxA for 20 min at various temperatures prior to substrate addition and further incubation at 37 °C showed that His-MxA resisted temperatures of up to 45 °C quite well but was rapidly inactivated at temperatures of 50 °C or higher (data not shown). To determine the enzymatic parameters of GTP hydrolysis by His-MxA, the substrate concentration was varied from 0.05 to 1.75 mM, and the reaction products were quantified (Fig. 1C). The K for GTP hydrolysis by His-MxA was 260 µM. Independent preparations of His-MxA varied slightly in activity; typically, they had specific GTPase activities of 50-350 nmol of GTP/min/mg of protein, corresponding to a turnover number of up to 27 min. Purified His-MxA could be stored for at least 2 weeks at -80 °C in buffer containing 10% glycerol without significant loss of GTPase activity.

To investigate further the substrate specificity of His-MxA, we performed competition experiments with ATP, GDP, and GTP analogs (GTPS and GMP-PNP) which have a cleavage-resistant bond between the - and -phosphate. At equimolar concentrations (100 µM GTP and 100 µM competitor), ATP and GDP were poor inhibitors of GTP hydrolysis by His-MxA. By contrast, GTPS and GMP-PNP were very efficient inhibitors (Fig. 2A). At 10-fold molar excess of competitor (100 µM GTP and 1000 µM competitor) GDP also showed an inhibitory effect, whereas ATP remained ineffective (Fig. 2B). These results indicated that His-MxA has a higher affinity for GTP than GDP.


Figure 2: Inhibition of His-MxA GTPase by various nucleotides. The GTP hydrolysis rate of His-MxA (0.1 µg/µl) was analyzed in the presence of 100 µM GTP and ATP, GDP, GMP-PNP, or GTPS at either 100 µM (panel A) or 1 mM (panel B). Reaction products were analyzed by polyethyleneimine-cellulose thin layer chromatography and autoradiography.



Purified His-MxA Has an Empty Nucleotide Binding Pocket

HPLC analysis was performed to identify any nucleotides that might have remained bound to His-MxA during the purification procedure. Proteins are rapidly denatured under these experimental conditions, and the released nucleotide can be detected spectrophotometrically (30, 31) . No GTP and GDP signals were observed when a 10-µl sample of a 226 µM solution of His-MxA was applied to the column (Fig. 3A). Control runs with 10 µl of a 20 µM solution of GTP and GDP gave strong signals, demonstrating that this assay system indeed allowed detection of guanine nucleotides at very high sensitivity (Fig. 3B). Mixing experiments showed that guanine nucleotides could be detected with similar sensitivity in solutions containing 226 µM ovalbumin (data not shown). We concluded from these experiments that more than 99% of the purified His-MxA molecules had an empty nucleotide binding pocket.


Figure 3: Purified His-MxA is free of guanine nucleotides. Panel A, a 10-µl sample of purified His-MxA (17 mg/ml = 226 µM) was applied to a HPLC column to detect guanine nucleotides that might have copurified. Panel B, a 10-µl sample of a premixed mixture containing 20 µM GDP and 20 µM GTP served as a standard. The chromatograms were monitored at 252 nm. The identity of the nucleotide peaks and the retention times are indicated.



Determination of Binding Constants for GTP and GDP

To gain proper information on the guanine nucleotide binding properties of His-MxA, we performed binding studies in solution with fluorescent nucleotides. A first series of fluorescence titration experiments was performed with mant-GMP-PNP, a fluorescent-modified nonhydrolyzable GTP analog. Adding increasing amounts of His-MxA to a 0.2 µM solution of mant-GMP-PNP resulted in a dose-dependent increase of fluorescence. The maximal change in fluorescence was 2.5-fold. The different data points could be fitted to a hyperbolic curve, which describes the binding of mant-GMP-PNP to His-MxA (Fig. 4). To determine the active binding sites in our preparation of His-MxA, mant-GMP-PNP was kept at 9 µM, and increasing amounts of His-MxA were added. Fluorescence increased in a linear fashion until saturation was reached at 14.3 µM His-MxA (Fig. 4, inset), indicating that about 62% of the His-MxA molecules participated in nucleotide binding. After correcting the results of the fluorescence titration experiment for active binding sites, we calculated the dissociation constant K of His-MxA for mant-GMP-PNP to be 0.75 µM.


Figure 4: Determination of the dissociation constant K for the His-MxAmant-GMP-PNP complex by fluorescence titration. The concentration of mant-GMP-PNP was 0.2 µM. The concentration of His-MxA was corrected for active sites (see inset). The data points were fitted as described under ``Experimental Procedures.'' The calculated K was 0.75 µM. Inset, active binding sites of His-MxA. His-MxA was added to saturation to a 9 µM solution of mant-GMP-PNP. The number of active sites was 62%.



To learn about the dynamics of the binding reaction, we performed stopped-flow experiments with His-MxA and mant-GMP-PNP. Increasing concentrations of mant-GMP-PNP were mixed with His-MxA ([His-MxA] 1/10 of [mant-GMP-PNP]), and fluorescence was monitored instantly. Under these conditions pseudo-first-order kinetics of the binding reaction can be assumed. The observed association rate k resulted from fitting the fluorescence change under various conditions to an exponential curve. The k values were plotted against the mant-GMP-PNP concentration and fitted to a straight line (Fig. 5A). The values for k increased linearly with the mant-GMP-PNP concentration up to 100 µM. The calculated association rate constant K of His-MxA for mant-GMP-PNP was 22,200 M s. K could not be determined from this linear fit.


Figure 5: Association and dissociation rates for His-MxA and mant-GMP-PNP measured by stopped-flow fluorometry. Panel A, concentration dependence of the pseudo-first-order rate constant for mant-GMP-PNP association. His-MxA ([His-MxA] 1/10 of [mant-GMP-PNP]) and increasing amounts of mant-GMP-PNP were mixed, fluorescence was recorded, and the k values were calculated. After fitting the various data points to a straight line, the calculated K for mant-GMP-PNP was 22,200 M s. Panel B, time course of displacement of mant-GMP-PNP from His-MxAmant-GMP-PNP (1 µM each) by 1 mM GDP. The calculated K for mant-GMP-PNP was 0.012 s, the deduced K was 0.54 µM.



To determine the rate of dissociation k for mant-GMP-PNP, His-MxA and mant-GMP-PNP at 1 µM each were allowed to reach an equilibrium (about 2 min) before unlabeled GDP was added to a final concentration of 1 mM and the change in fluorescence was recorded. Fluorescence decreased rather rapidly and reached a plateau after about 400 s (Fig. 5B). The calculated dissociation rate constant K for mant-GMP-PNP was 0.012 s. The K for mant-GMP-PNP determined by this method was 0.54 µM, which is in good agreement with the value deduced from the titration experiment described above. In another experiment, 5 mM unlabeled GTP was used as competitor. Under these conditions, the calculated K was again 0.012 s (data not shown).

To determine the binding kinetics and binding constant of His-MxA for GDP, stopped-flow experiments were performed with mant-GDP. The results (Fig. 6) permitted calculation of the kinetic parameters of His-MxA for mant-GDP: K was 430,000 M s, K was 8.5 s, and the deduced K was 20 µM.


Figure 6: Association and dissociation rates of mant-GDP determined by stopped-flow fluorometry. The concentration dependence of the pseudo-first-order rate constant for association of mant-GDP and His-MxA is shown. The circles indicate data points from experiments with mant-GDP at more than 10-fold molar excess over His-MxA. The His-MxA concentration was corrected for active sites, and the data points were fitted to a straight line. The calculated K for mant-GDP was 430,000 M s; the K was 8.5 s, and the K was 20 µM.



Since the mant modification can influence the binding properties of nucleotides (33, 34) , we determined the K of His-MxA for unlabeled guanine nucleotides by competitive binding assays. Samples of His-MxA (2 µM) and mant-GMP-PNP (1 µM) were mixed, and while increasing concentrations of either GTPS, GMP-PNP, or GDP were added, the fluorescence changes of the mixtures were monitored. This procedure made it possible to determine the concentrations of the various competitors required for half-maximal reduction of the fluorescence signals. After correcting for active binding sites, the dissociation constants K for GTPS, GMP-PNP, and GDP were calculated to be 1.7, 8, and 100 µM, respectively (). Determining the K for GTP was complicated by the fact that it is rapidly hydrolyzed by His-MxA. To minimize interference from hydrolysis, we chose to restrict our measurements to very early times after the addition of the competitor (less than 3 min). When the data points from the different fluorescence measurements were analyzed, the dissociation constant K of GTP was calculated to be 20 µM (). These results showed that unlabeled GMP-PNP and GDP bound less well to His-MxA than their mant-labeled counterparts. Furthermore, these results demonstrated that His-MxA has a high binding preference for GTP over GDP.


DISCUSSION

We have shown previously that recombinant His-MxA retains GTPase activity during purification from bacteria that express an appropriate cDNA construct (25, 26) . Purified His-MxA was used successfully to inhibit the transcriptase of vesicular stomatitis (26) and influenza A virus (27) in vitro, demonstrating that it retained antiviral activity. Thus, it was of interest to characterize this material in more detail and to determine the biochemical parameters of the MxA-associated GTPase activity. We have shown here that His-MxA is a GTPase that requires very high substrate concentrations for half-maximal reaction velocity. It further exhibits rather low affinity for GTP and extremely low affinity for GDP. These biochemical parameters stress the unique character of Mx proteins and justify a separate classification in the GTPase superfamily.

The GTP turnover number of our most active preparations of His-MxA was 27 min, and the K of the GTP hydrolysis reaction was 260 µM. These values agree quite well with those for natural MxA purified by immunoprecipitation from interferon-induced human fibroblasts (28) . However, the turnover number of recombinant MxA was about 3-fold lower than that of immobilized antibody-complexed natural MxA, which showed a GTP conversion rate of 70 min(28) . The lower activity of the recombinant protein could be due to a partial inactivation of His-MxA during the more laborious purification procedure. Alternatively, the higher GTPase activity of natural MxA (28) may have resulted from a stimulation by the antibody used for immunoprecipitation. We recently observed that the GTPase activity of purified His-MxA can indeed be stimulated 3-4-fold by adding equimolar amounts of monoclonal antibody 2C12.() A third possibility is that the immunoprecipitates contained a cellular factor that stimulated GTPase activity.

HPLC analysis showed that purified His-MxA had an empty nucleotide binding pocket. Bound nucleotides thus seem to dissociate quickly from MxA during purification in nucleotide-free buffer. This view was confirmed in direct binding experiments with guanine nucleotides carrying a modification that allows the monitoring of the binding events by fluorescence techniques (32) . The K of His-MxA for the fluorescent nonhydrolyzable GTP analog mant-GMP-PNP was 0.54-0.75 µM, and the half-life of the His-MxAmant-GMP-PNP complex was about 80 s. His-MxA showed 30-40-fold lower affinity for mant-GDP, and the half-life of the His-MxAmant-GDP complex was only about 0.1 s. Competition experiments with unmodified nucleotides showed that the complexes were not destabilized by the mant modification. In fact, His-MxA bound the natural nucleotides even less well than their modified counterparts; the K of His-MxA for unmodified GTP was about 20 µM, and the K for unmodified GDP was about 100 µM. These values differ dramatically from those of p21 and other small molecular weight GTPases which have K values for GTP and GDP in the picomolar range (33) .

The results of our binding experiments do not agree with a recent study (29) in which the guanine nucleotide binding activity of recombinant MxA protein was assessed by filter binding assays. The important difference is that our analysis showed that MxA has about 5-fold higher affinity for GTP than for GDP, whereas the former study suggested the opposite. Obviously, the filter binding assay is less suitable for measuring low affinity interactions between protein and nucleotide than binding experiments in solution.

The nucleotide binding studies presented here showed that about two-thirds of the His-MxA molecules in our preparations participated actively in the GTP binding reaction. Since gel filtration experiments demonstrated that the bulk of His-MxA was present in form of high molecular weight oligomers, these studies suggest that most of the MxA monomers in the large complexes remained functional.

The biochemical parameters of the MxA-associated GTPase which we have determined here allow us to describe the GTPase cycle of MxA in vivo. The concentration of GTP in eukaryotic cells was estimated to be approximately 100 µM(4) , which is above the K of MxA for this nucleotide (20 µM). The intracellular concentration of GDP is severalfold lower (4) and thus far below the K of MxA for GDP (100 µM). Thus, most of the MxA molecules may be complexed with GTP under physiological conditions. Our results further predict that once MxA-bound GTP was hydrolyzed, the newly formed GDP was released very rapidly, and the empty nucleotide binding pocket of MxA was ready to accommodate a new GTP molecule. It is important to note that the biochemical parameters of MxA predict that the GTPase cycle proceeds without auxiliary factors. In particular, it is unnecessary to postulate a nucleotide exchange factor for MxA. Nonetheless, to prevent futile degradation of GTP, the GTPase activity of Mx proteins may be regulated in vivo. Our findings that the in vitro GTPase activity of purified His-MxA could be stimulated 3-4-fold by a monoclonal antibody suggest that control is indeed possible at this level. Mx-associated proteins that might serve regulatory functions have not been identified to date. In this context it is of interest to note that the in vitro and in vivo GTPase activity of dynamin, a phosphoprotein with homology to Mx, can be stimulated by proteins that carry SH3 domains (35, 36, 37) and by several other means (12, 14, 24, 38) .

GTPases are prime candidates for molecular switches, as they can assume distinct conformations in the GTP-bound and GDP-bound states (4). The GTP-bound form typically represents the active state of regulatory GTPases, whereas the GDP-bound form represents the inactive state. Assuming that the biological activity of Mx proteins is also regulated by guanine nucleotides, the biochemical parameters that we have determined here for His-MxA would suggest that the MxAGDP complex cannot serve as a switch position because it is extremely unstable under physiological conditions. Our data suggest that most of the MxA molecules exist in the GTP-bound state. The empty state rather than the GDP-bound state may represent the alternative conformation in the case of Mx proteins.

  
Table: K of His-MxA for various nonfluorescent nucleotides

His-MxA (2 µM) was allowed to react with fluorescent mant-GMP-PNP (1 µM) before increasing concentrations (from 1 µM up to 1.5 mM) of the indicated competitor nucleotides were added. The competitor concentrations required for half-maximal reduction of fluorescence were recorded, and the K values were calculated.



FOOTNOTES

*
This work was supported by a grant from the Deutsche Forschungsgemeinschaft. 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.

§
To whom correspondence should be addressed: Dept. of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79008 Freiburg, Germany. Tel.: 49-761-203-6579; Fax: 49-761-203-6562.

Pavlovic, J., Arzet, H. A., Hefti, H. P., Frese, M., Rost, D., Ernst, B., Kolb, E., Staeheli, P., and Haller, O. (1995) J. Virol. 69, in press.

The abbreviations used are: His-MxA, histidine-tagged MxA; AMP-PNP, 5`-adenylyl-,-imidodiphosphate; HPLC, high pressure liquid chromatography; GMP-PNP, 5`-guanylyl-,-imidodiphosphate; mant, N-methylanthraniloyl.

M. F. Richter, unpublished results.


ACKNOWLEDGEMENTS

We thank Jovan Pavlovic for numerous helpful discussions at an early stage of this work.


REFERENCES
  1. Bourne, H. R., Sanders, D. A., and McCormick, F.(1990) Nature 348, 125-132 [CrossRef][Medline] [Order article via Infotrieve]
  2. Sprinzl, M.(1994) Trends Biochem. Sci. 19, 246-250
  3. Rothman, J. E., and Orci, L.(1992) Nature 355, 409-415 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bourne, H. R., Sanders, D. A., and McCormick, F.(1991) Nature 349, 117-127 [CrossRef][Medline] [Order article via Infotrieve]
  5. Melancon, P.(1993) Curr. Biol. 3, 230-233 [Medline] [Order article via Infotrieve]
  6. Wittinghofer, A., and Pai, E. F.(1991) Trends Biochem. Sci. 16, 382-387 [CrossRef][Medline] [Order article via Infotrieve]
  7. Fischer von Mollard, G., Stahl, B., Li, C., Sudhof, T. C., and Jahn, R. (1994) Trends Biochem. Sci. 19, 164-168 [CrossRef][Medline] [Order article via Infotrieve]
  8. Moore, M. S., and Blobel, G.(1994) Trends Biochem. Sci. 19, 211-216 [CrossRef][Medline] [Order article via Infotrieve]
  9. Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168 [CrossRef][Medline] [Order article via Infotrieve]
  10. Miller, J. D., Wilhelm, H., Gierasch, L., Gilmore, R., and Walter, P. (1993) Nature 366, 351-354 [CrossRef][Medline] [Order article via Infotrieve]
  11. Markby, D. W., Onrust, R., and Bourne, H. R.(1993) Science 262, 1895-1901 [Medline] [Order article via Infotrieve]
  12. Shpetner, H. S., and Vallee, R. B.(1992) Nature 355, 733-735 [CrossRef][Medline] [Order article via Infotrieve]
  13. Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W., Poodry, C. A., Wadsworth, S. C., and Vallee, R. B.(1991) Nature 351, 583-586 [CrossRef][Medline] [Order article via Infotrieve]
  14. van der Bliek, A. M., and Meyerowitz, E. M.(1991) Nature 351, 411-414 [CrossRef][Medline] [Order article via Infotrieve]
  15. Collins, C. A.(1991) Trends Cell Biol. 1, 57-60 [Medline] [Order article via Infotrieve]
  16. Yeh, E., Driscoll, R., Coltrera, M., Olins, A., and Bloom, K.(1991) Nature 349, 713-715 [CrossRef][Medline] [Order article via Infotrieve]
  17. Vater, C. A., Raymond, C. K., Ekena, K., Howald-Stevenson, I., and Stevens, T. H.(1992) J. Cell Biol. 119, 773-786 [Abstract]
  18. Rothman, J. H., Raymond, C. K., Gilbert, T., O'Hara, P. J., and Stevens, T. H.(1990) Cell 61, 1063-1074 [Medline] [Order article via Infotrieve]
  19. Staeheli, P.(1990) Adv. Virus Res. 38, 147-200 [Medline] [Order article via Infotrieve]
  20. Pavlovic, J., Zurcher, T., Haller, O., and Staeheli, P.(1990) J. Virol. 64, 3370-3375 [Medline] [Order article via Infotrieve]
  21. Schnorr, J. J., Schneider-Schaulies, S., Simon Jodicke, A., Pavlovic, J., Horisberger, M. A., and ter Meulen, V.(1993) J. Virol. 67, 4760-4768 [Abstract]
  22. Schneider-Schaulies, S., Schneider-Schaulies, J., Schuster, A., Bayer, M., Pavlovic, J., and ter Meulen, V.(1994) J. Virol. 68, 6910-6917 [Abstract]
  23. Frese, M., Kochs, G., Meier-Dieter, U., Siebler, J., and Haller, O. (1995) J. Virol., 69, in press
  24. Robinson, P. J., Sontag, J. M., Liu, J. P., Fykse, E. M., Slaughter, C., McMahon, H., and Sudhof, T. C.(1993) Nature 365, 163-166 [CrossRef][Medline] [Order article via Infotrieve]
  25. Pitossi, F., Blank, A., Schröder, A., Schwarz, A., Hüssi, P., Schwemmle, M., Pavlovic, J., and Staeheli, P.(1993) J. Virol. 67, 6726-6732 [Abstract]
  26. Schwemmle, M., Weining, K. C., Richter, M. F., Schumacher, B., and Staeheli, P.(1995) Virology 206, 545-554 [Medline] [Order article via Infotrieve]
  27. Landis, H., Hefti, H. P., and Pavlovic, J.(1994) Virology, in press
  28. Horisberger, M. A.(1992) J. Virol. 66, 4705-4709 [Abstract]
  29. Melen, K., Ronni, T., Lotta, T., and Julkunen, I.(1994) J. Biol. Chem. 269, 2009-2015 [Abstract/Free Full Text]
  30. Tucker, J., Sczakiel, G., Feuerstein, J., John, J., Goody, R. S., and Wittinghofer, A.(1986) EMBO J. 5, 1351-1358 [Abstract]
  31. Feuerstein, J., Goody, R. S., and Wittinghofer, A.(1987) J. Biol. Chem. 262, 8455-8458 [Abstract/Free Full Text]
  32. Hiratsuka, T.(1983) Biochim. Biophys. Acta 742, 496-508 [Medline] [Order article via Infotrieve]
  33. John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A., and Goody, R. S.(1990) Biochemistry 29, 6058-6065 [Medline] [Order article via Infotrieve]
  34. Woodward, S. K. A., Eccleston, J. F., and Geeves, M. A.(1991) Biochemistry 30, 422-430 [Medline] [Order article via Infotrieve]
  35. Herskovits, J. S., Shpetner, H. S., Burgess, C. C., and Vallee, R. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11468-11472 [Abstract]
  36. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D.(1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  37. Trowbridge, I. S.(1993) Curr. Biol. 3, 773-775 [Medline] [Order article via Infotrieve]
  38. Tuma, P. L., Stachniak, M. C., and Collins, C. A.(1993) J. Biol. Chem. 268, 17240-17246 [Abstract/Free Full Text]

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