©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Unexpected Structural Requirements for GTPase Activity of the Interferon-induced MxA Protein(*)

Martin Schwemmle , Marc F. Richter , Christian Herrmann (1), Nicolas Nassar (1), Peter Staeheli (§)

From the (1) Abteilung Virologie, Institut für medinische 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 an interferon-induced 76-kDa GTPase that inhibits the multiplication of several RNA viruses. Deleting seven amino acids from the COOH terminus reduced the GTPase activity of purified MxA to 1.4%. MxA mutants with COOH-terminal deletions of 63 or more amino acids lost all ability to hydrolyze GTP and failed to bind guanine nucleotides. By contrast, an MxA deletion mutant consisting of 301 amino acids from the NH terminus and 87 amino acids from the COOH terminus retained about 9% of wild-type GTPase activity, underscoring the pivotal role of COOH-terminal sequences. Limited proteolysis of wild-type MxA with proteinase K resulted in two resistant polypeptides of 60 and 10 kDa, respectively, which copurified as a stable complex. The p60-p10 complex exhibited high GTPase activity, suggesting that it included all MxA domains required for this biochemical activity. Sequencing revealed that the NH terminus of the 60-kDa polypeptide mapped to leucine 41 and the NH terminus of the 10-kDa polypeptide to glutamine 564 of the MxA sequence. Based on these results we propose a model that suggests that the GTP-binding consensus element located in the NH-terminal half of MxA is held in an active conformation by strong physical interactions with amino acids from the COOH-terminal region.


INTRODUCTION

The interferon-induced human MxA protein is a GTPase of 76-kDa molecular mass which exhibits antiviral activity (1, 2, 3, 4, 6) .() The loss of GTP binding due to mutations in the GTP-binding element of MxA was accompanied by a loss of antiviral activity (7) , suggesting that nucleotide binding is important for function. Recombinant histidine-tagged MxA protein (His-MxA)() purified from Escherichia coli retains high GTPase activity; up to 27 mol of GTP were hydrolyzed per mol of His-MxA/min (8) . His-MxA shows low affinity for GTP (8) , and the K for the His-MxA-catalyzed GTP hydrolysis reaction is characteristically high (8). Natural and recombinant His-MxA form high molecular weight oligomers, a characteristic of unknown significance. His-MxA purified from E. coli retains antiviral activity; it blocked in vitro transcription of vesicular stomatitis virus (9) and influenza A virus (10) . Inhibition of viral polymerase activity by His-MxA was observed irrespective of whether the reaction mixture contained GTP or nonhydrolyzable GTP analogs (9, 10) , suggesting that binding rather than hydrolysis of GTP is of functional importance.

Little is known about the functional domains of MxA. A domain that determines antiviral specificity is located near the COOH terminus; MxA with a single amino acid substitution at position 645 was active against influenza A virus, but unlike wild-type MxA, the mutant protein failed to inhibit vesicular stomatitis virus in vivo(11) and in vitro(9) . GTPase activity of His-MxA was not affected by this mutation (9) . Mutation analysis of the mouse Mx1 protein (12, 13) showed that COOH-terminal deletions and most point mutations in various regions of Mx1 resulted in a loss of antiviral activity, suggesting a complex organization of the functional domains.

While studying the inhibitory effect of His-MxA on vesicular stomatitis virus transcription in vitro(9) , we observed that a His-MxA mutant that lacks the COOH-terminal 300 amino acids had lost GTPase activity. This result suggested that the NH-terminal fragment that harbors the tripartite GTP-binding consensus element (14, 15) requires sequences from the COOH terminus to form a functional GTPase. We have now studied the influence of COOH-terminal sequences of MxA for GTPase activity in more detail. We provide genetic and biochemical evidence that the COOH-terminal 99 amino acids are required for GTPase activity, whereas sequences located between the GTP-binding consensus element and the COOH-terminal domain are dispensable. Mild proteolysis of His-MxA yielded a large NH-terminal and a small COOH-terminal polypeptide that formed a stable GTPase-active complex, suggesting that the COOH terminus folds back and interacts with the GTP-binding consensus element to form the active center of the enzyme.


EXPERIMENTAL PROCEDURES

Plasmid Constructions

All mutant constructs were derived from plasmid pHis-MxA (7, 8, 9) , a derivative of the E. coli expression vector pQE 9 (Diagen, Hilden, Germany) which contains MxA cDNA. The construct pHis-MxA(1-301), pHis-MxA(1-362), pHis-MxA(1-599), and pHis-MxA(1-655) were generated by digesting full-length pHis-MxA with HindIII and either BglII, SalI, BclI, or NarI, blunting the ends with Klenow polymerase, and re-ligating the products. Because of this cloning procedure, the mutant MxA proteins had extra amino acids at their COOH termini. The extra sequence of His-MxA(1-301) and His-MxA(1-599) was Ser-Leu-Ile-Ser, of His-MxA(1-655) Gln-Pro-Asn, and of His-MxA(1-362) Glu-Ala. The construct pHis-MxA(301-576) was generated by cutting pHis-MxA with BglII to release a 725-nucleotide fragment and re-ligating the products.

Purification of Histidine-tagged Wild-type and Mutant MxA Proteins

Histidine-tagged wild-type and mutant MxA proteins were purified from E. coli as described (7, 8, 9) .

Purification of Proteinase K-resistant Fragments of His-MxA

Purified His-MxA (1-2 mg/ml) was incubated with 0.004 mg/ml proteinase K (Serva, Heidelberg, Germany) for 20 min at room temperature in buffer A (50 µM Tris-HCl, pH 8.0, 10% glycerol, 1 µM 2-mercaptoethanol 100 mM NaCl and 11.6 mM MgCl). Complete conversion of His-MxA to the p60-p10 complex was verified by running treated and untreated samples on SDS-PAGE gels and staining with Coomassie Blue. 200-µl samples of proteinase K-treated His-MxA were loaded on a Superose 12 HR10/30 gel filtration column (Pharmacia, Freiburg, Germany) equilibrated with buffer B (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl, 10% glycerol, and 1 mM 2-mercaptoethanol), and fractions of 0.5 ml were collected. Most of the p60-p10 complex was found in the void volume. This material was applied to a HR5/5 Mono Q chromatography column (Pharmacia) equilibrated with buffer B containing 5 mM 2-mercaptoethanol. The p60-p10 complex eluted at 350 mM NaCl.

For large scale purifications, 1 ml of proteinase K-treated His-MxA (6-8 mg/ml) was subjected to a Hi Load 16/60 Superdex 200 HR preparative grade gel filtration column (Pharmacia) equilibrated with glycerol-free buffer B. Column fractions between void volume (>600 kDa) and 400 kDa were pooled and purified by Mono Q chromatography as described above. Yields of p60-p10 complex were about 20-40% of the His-MxA starting material.

Protein Sequencing

The proteinase K-resistant 60-kDa and 10-kDa polypeptides were recovered from 10% and 20% SDS-PAGE gels, respectively, by electroblotting onto a polyvinylidene difluoride membrane. Sequencing from the NH terminus was performed with an Applied Biosystems 477A Protein Sequencer.

GTPase Assays

GTPase assays were performed as described (8, 9) . Briefly, 0.1-0.2 mg/ml purified wild-type or mutant His-MxA was incubated at 37 °C in buffer C (50 mM Tris-HCl, pH 8.0, 5 mM MgCl, 100 mM KCl, 10% glycerol, 0.1 mM dithiothreitol, 100 nM AMP-PNP, 13 nM [-P]GTP (3,000 Ci/mmol), and 1 mM GTP). Where indicated, a lower GTP concentration was used. After stopping reactions with 0.5% SDS and 2 mM EDTA, samples were spotted onto polyethylenimine-cellulose thin layer chromatography plates (MN300, Macherey and Nagel, Düren, Germany), and the nucleotides were separated in a solution containing 1 M LiCl and 1 M acetic acid. The dried plates were exposed to x-ray films. The signals were quantified using a digital autoradiograph LB286 (Berthold, Wildbad, Germany).

Determination of Dissociation Constants

Determination of the dissociation constants K of proteinase K-treated His-MxA and His-MxA(1-362) for the fluorescent nonhydrolyzable GTP analog mant-GMP-PNP was done as described for wild-type MxA (8) .

Estimating the Degree of His-MxA Oligomerization

The degree of oligomerization was estimated by performing gel filtration experiments. 500-µl samples of Mono Q-purified wild-type or mutant His-MxA (at about 1 mg/ml) were loaded on a Hi Load 16/6 Superdex 200 HR preparative grade gel filtration column (S-200) (Pharmacia) in buffer B at a flow rate of 1 ml/min, and fractions of 1.4 ml were collected. The elution profiles were analyzed by running samples of each second column fraction on 10% SDS-PAGE gels and staining with Coomassie Blue.


RESULTS

COOH-terminal Deletions Destroy GTPase Activity of MxA

While studying the effect of MxA on the in vitro transcription of vesicular stomatitis virus, we observed that the COOH-terminally truncated mutant His-MxA(1-362) lacked measurable GTPase activity (9) . To investigate the importance of COOH-terminal sequences for GTPase activity in more detail, we created E. coli expression constructs for a series of MxA mutants with COOH-terminal deletions of variable lengths (Fig. 1). The shortest deletion was 7 amino acids in His-MxA(1-655), the longest deletion was 361 amino acids in His-MxA(1-301). Because of the cloning procedure, all mutant proteins contained two to four foreign amino acids at the COOH terminus. Since wild-type and mutant His-MxA carried a histidine tag at the NH terminus, convenient purification from bacterial lysates was possible by a single round of nickel chelate agarose chromatography. We have shown previously that this procedure yields MxA of acceptable purity and high specific GTPase activity (7, 8, 9) . Several independent preparations of the mutants His-MxA(1-301), His-MxA(1-362), and His-MxA(1-599) lacked measurable GTPase activity, whereas preparations of His-MxA(1-655) showed very low GTPase activity (Fig. 1).


Figure 1: His-MxA variants and their biochemical properties. Panel A, several independent preparations of wild-type and the indicated mutant MxA proteins were produced in E. coli, purified, and tested for GTPase activity. After partial purification by nickel chelate affinity chromatography, the preparations were classified as having high (+++), moderate (+), weak (+/-), or no (-) GTPase activity. Selected preparations of the various MxA mutants were then purified by Mono Q column chromatography, and their specific GTPase activities were determined. Activities below 0.04 mol of GTP/min/mol could not be measured reliably under our experimental conditions. For some MxA variants, the K for GTP hydrolysis and the K for the fluorescent GTP analog mant-GMP-PNP were determined. n.d., not determined. The black bars numbered I-III in the drawings indicate the position of the tripartite GTP-binding consensus element. *, to determine the K of His-MxA(1-362) for mant-GMP-PNP, we added increasing amounts of protein (from 1 to 25 µM) to a solution of 1.5 µM mant-GMP-PNP. As no specific signals were observed under these conditions, this mutant has either no affinity for mant-GMP-PNP, or its K is higher than 100 µM. Panel B, wild-type His-MxA was subjected to mild degradation with proteinase K. The resulting complex consisting of the two indicated fragments p60 and p10 was purified, and its biochemical parameters were determined.



To measure the low GTPase activities more accurately, we purified selected preparations of the various MxA mutants by Mono Q column chromatography. Like wild-type His-MxA, the mutant proteins eluted from the Mono Q column between 250 and 350 mM NaCl. Analysis by SDS-PAGE revealed that mutants His-MxA(1-301) and His-MxA(1-362) were about 90% pure as judged by Coomassie Blue staining, whereas the other mutants were only about 50% pure (Fig. 2). The prominent contaminant at about 30 kDa presumably represents an NH-terminal fragment of His-MxA because it is recognized by an antiserum to full-length MxA but not by an antiserum to a COOH-terminal fragment that comprises amino acids 301 to 662 (data not shown). Purification by Mono Q chromatography of wild-type His-MxA had little effect on its specific GTPase activity, which was 11.4 mol of GTP/min/mol (Fig. 1). The Mono Q-purified mutants His-MxA(1-301), His-MxA(1-362), and His-MxA(1-599) failed to convert GTP to GDP at a rate that was high enough for detection under our assay conditions (<0.04 mol of GTP/min/mol) (Fig. 1). This picture did not change when the substrate concentration was varied from 0.1 to 1.5 mM (data not shown). The specific activity of His-MxA(1-655) was 0.15 mol of GTP/min/mol (Fig. 1). Mixing experiments showed that the COOH-terminal His-MxA mutants did not influence the GTP hydrolysis rate of wild-type His-MxA, demonstrating that the low GTPase activity in the former preparations was not due to the presence of inhibitory contaminants (data not shown).


Figure 2: Purity of wild-type (wt) and mutant His-MxA proteins. Samples (4 µg/lane) of the various His-MxA preparations after nickel chelate affinity chromatography and fractionation on Mono Q were run on a 10% SDS-PAGE gel and stained with Coomassie Blue. The expected gel positions of the various MxA proteins are indicated by arrows.



As purified wild-type His-MxA has a high tendency to form oligomers (8), we tested whether our inactive MxA mutants would show a different behavior. Running samples of the various His-MxA mutants on an S-200 gel filtration column revealed that they were all present as oligomers; the loaded material eluted mostly with the column void volume (data not shown), indicating that the oligomers were 600 kDa or larger.

Since the various MxA deletion mutants contained the tripartite GTP-binding consensus element, which is located in the NH-terminal half of MxA (Fig. 1), it was of interest to test whether they would still bind GTP. Since His-MxA(1-362) could be purified most easily, we chose this mutant for binding studies that were performed with the fluorescence-labeled nonhydrolyzable GTP analog mant-GMP-PNP (8) . Using this assay, the dissociation constant of wild-type His-MxA for mant-GMP-PNP was determined to be 0.75 µM (Fig. 1). However, we failed to demonstrate specific binding of fluorescent mant-GMP-PNP to His-MxA(1-362) under identical assay conditions, indicating that the K was higher than 100 µM. This result suggested that COOH-terminal sequences of MxA are required for both binding and hydrolysis of GTP.

A Deletion Mutant with Intact COOH Terminus Exhibits GTPase Activity

To challenge the hypothesis that sequences from near the COOH terminus of MxA are required for GTP hydrolysis, we generated the expression construct pHis-MxA(301-576), which codes for an MxA variant with intact NH and COOH termini but which lacks the 275 amino acids between positions 301 and 576 (Fig. 1). His-MxA(301-576) could be purified fairly well from bacterial lysates (Fig. 2) and had a specific GTPase activity of 0.98 mol of GTP/min/mol, which corresponds to about 9% of wild-type activity. A more detailed examination showed that the K for the GTP hydrolysis reaction catalyzed by His-MxA(301-576) was 1,150 µM (Fig. 1). The K of wild-type His-MxA for this reaction was 260 µM.

Treating MxA with Proteinase K Yields Two Resistant Polypeptides of 60 and 10 kDa Which Form a GTPase-active Complex

Proteins are often composed of functional domains that are connected by linkers. The linkers are usually more accessible to proteolytic degradation than the compact structures of the domains. Mild treatment of purified wild-type His-MxA with proteinase K resulted in rapid degradation of the full-length protein and accumulation of a prominent 60-kDa fragment (p60) which could be visualized by 10% SDS-PAGE (Fig. 3A). Interestingly, proteinase K-treated His-MxA retained GTPase activity (Fig. 3B), indicating that the catalytic site remained functional. Conversion of His-MxA to p60 was completed within about 5 min, and the p60 polypeptide resisted further degradation by proteinase K for up to 120 min (data not shown). The yields of proteinase K-resistant fragment varied between preparations of His-MxA and were negatively affected by prolonged storage at -80 °C and repeated cycles of freezing and thawing. Analysis of proteinase K-treated His-MxA by 20% SDS-PAGE revealed the presence of several small protein fragments, but their significance was unclear. To examine whether one of these polypeptides was complexed with p60, proteinase K-treated His-MxA was subjected to gel filtration on a Superose 12 column, and samples of the various fractions were analyzed for polypeptide content and GTPase activity. The bulk of p60 eluted with the void volume (Fig. 4, lanes 2-4), indicating that it formed oligomers like full-length His-MxA. Column fractions containing p60 showed approximately equimolar amounts of a 10-kDa polypeptide (p10), suggesting that the two fragments formed a complex. The column fractions that exhibited high GTPase activity contained both p60 and p10 (Fig. 4B), suggesting that the p60-p10 complex was the active entity. Further purification of the p60-p10 complex was achieved by applying pooled gel filtration fractions (Fig. 5, lane 1) containing p60-p10 to Mono Q chromatography. Both the p60-p10 complex and GTPase activity eluted from this column at about 350 mM NaCl (Fig. 5, lanes 2-5). The complex showed a specific GTPase activity of 11 mol of GTP/min/mol, its K for GTP hydrolysis was 330 µM, and its K for mant-GMP-PNP was 2 µM (Fig. 1). These values were almost identical to those of full-length His-MxA, suggesting that proteinase K treatment did not affect the catalytic site and that the association between p60 and p10 was similar to wild-type MxA.


Figure 3: Mild digestion of His-MxA with proteinase K has little effect on GTPase activity. Panel A, samples of Mono Q-purified His-MxA were analyzed by 10% SDS-PAGE before (lane 1) or after (lane 2) incubation with proteinase K. Panel B, samples of corresponding lanes were assayed for GTPase activity by incubating with radiolabeled GTP and determining the degree of hydrolysis to GDP by thin layer chromatography and autoradiography.




Figure 4: A complex of two polypeptides results from digestion of His-MxA with proteinase K. Proteinase K-treated His-MxA was subjected to gel filtration on a Superose 12 column, and fractions of 500 µl were collected. Panel A, samples were analyzed by 20% SDS-PAGE and Coomassie Blue staining. The gel filtration properties of blue dextran 2000 that was found in the void volume of this column (>300 kDa) and bovine serum albumin (BSA, 67 kDa) are indicated. Panel B, samples of corresponding lanes were assayed for GTPase activity by incubating with radiolabeled GTP and determining the degree of hydrolysis to GDP by thin layer chromatography and autoradiography.




Figure 5: Purification of the p60-p10 complex. Panel A, His-MxA treated with proteinase K was subjected to gel filtration, and the pooled peak fractions (lane 1) were loaded on a Mono Q column. Bound proteins were eluted with a linear salt gradient. Fractions around 350 mM NaCl (lanes 2-5) were analyzed by 20% SDS-PAGE, and proteins present in these fractions were visualized by silver staining. Panel B, samples of corresponding lanes were assayed for GTPase activity by incubating with radiolabeled GTP and determining the degree of hydrolysis to GDP by thin layer chromatography and autoradiography.



To characterize the p60-p10 complex further, the two polypeptides were separated by preparative SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to NH-terminal sequencing. The NH-terminal sequence of p60 was LCSQYNN, which corresponds to positions 41-47 of MxA (Fig. 6); the NH-terminal sequence of p10 was QSSSATD, which corresponds to positions 564-570. Thus, proteinase K treatment removed the 40 nonconserved NH-terminal amino acids of MxA as well as a small internal fragment. The internal cleavage site maps to a region of MxA (positions 555-575) which is poorly conserved in Mx proteins from mammals and birds (16) . These results agreed well with the concept that had emerged from the MxA deletion analysis, which suggested intramolecular interactions of COOH- and NH-terminal sequences.


Figure 6: NH-terminal sequences of proteinase K-resistant fragments p60 and p10 and relationship to full-length MxA. After partial digestion of His-MxA with proteinase K, the resistant polypeptides p60 and p10 were gel purified and sequenced from their NH termini. Derived sequence information (indicated in capital letters, single-letter code) allowed to map the two fragments to MxA regions with high sequence conservation. Dotted areas mark two regions of MxA which were identified as highly variable by previous sequence comparisons (16).




DISCUSSION

The principal conclusion from the experiments presented here is that binding and hydrolysis of GTP by the interferon-induced MxA protein require both an intact GTP-binding consensus element and an intact COOH terminus. Deletion mutants of MxA that lacked 7 or more COOH-terminal amino acids exhibited greatly reduced GTPase activity, whereas a mutant with intact NH and COOH termini but lacking 275 internal amino acids retained a high degree of activity. Furthermore, biochemical analysis showed that sequences from the NH-terminal half of MxA which include the tripartite GTP-binding consensus element are in physical contact with amino acids from near the COOH terminus. Thus, the structural requirements for GTP binding and GTP hydrolysis by MxA are without precedent. Our results suggest that the GTP binding pocket of MxA has a structure that could be distinct from that of p21, elongation factor Tu, and other well known GTPases (17, 18, 19, 20) . They may explain the unique biochemical parameters of MxA: low affinity for GTP (K 20 µM) and even lower affinity for GDP (K 100 µM), the need for very high substrate concentrations for high rates of GTP hydrolysis (k 27 min, K 260 µM), and no need of accessory factors for performing multiple cycles of GTP hydrolysis (8) .

Our findings that even small COOH-terminal deletions had a strong negative effect on the GTPase activity of MxA was unexpected in the light of earlier findings with the Mx-related protein Vps-1p of yeasts. Radiolabeled GTP bound similarly well to full-length Vps-1p and certain NH-terminal Vps-1p fragments (21) , suggesting that a relatively small region that comprises the tripartite GTP-binding consensus element is necessary and sufficient for binding of GTP by Vps-1p. Since our experiments with deletion mutant His-MxA(1-362) showed that COOH-terminal sequences are not only required for hydrolysis but also for binding of GTP to MxA, it appears that the nucleotide binding pockets of Vps-1p and MxA have also quite different structures despite sequence similarity (22) . Unfortunately, a direct comparison of the Vps-1p and MxA mutant studies is not possible because no information is available on the GTP hydrolysis activities of the various Vps-1p mutants.

Mild degradation of purified His-MxA with proteinase K yielded valuable information regarding the tertiary structure of this GTPase. Protease treatment has been applied to remove poorly structured amino acid sequences from a variety of proteins, including the G protein G(23, 24) , elongation factor Tu (25, 26) , and methionyl-tRNA synthetase (27) . Typically, the resulting protease-resistant polypeptides were easier to crystallize than the starting material, presumably because their structures were more compact. In the case of MxA, two protease-resistant fragments (p60 and p10) were identified which corresponded almost exactly to Mx regions previously identified as highly conserved among mammals and birds (16) . Since the two fragments copurified as a stable complex during gel filtration and ion exchange chromatography, they must interact very strongly with each other. It is unknown at present which amino acids are actively participating in this interaction. Mutational analysis will be necessary to determine whether the putative leucine zipper motif located near the COOH termini of all Mx proteins (28) is involved. Our results suggest that a compact structure (p10) that consists of the 99 COOH-terminal amino acids folds back to interact with the body (p60) of the MxA molecule (Fig. 7). The short stretch of nonconserved amino acids located between p10 and p60 may serve as a hinge. We believe that the alternative possibility that p10 of a first MxA molecules interacts with p60 of a second MxA molecule is less likely. Such a scenario would be compatible with the observation that MxA forms high molecular weight oligomers. However, proteinase K should then degrade the MxA oligomers to monomers, which was clearly not the case; the p60-p10 complex purified as a high molecular weight entity (Fig. 4). Since all MxA variants described in this study retained the ability to form high molecular weight oligomers, it seems that oligomerization of MxA is mediated by sequences located between amino acids 41 and 301. This view is in agreement with results from experiments with the mouse Mx1 protein which defined a self-assembly motif in the NH-terminal moiety near the GTP-binding element (5) .


Figure 7: Two-domain model for the MxA-associated GTPase activity. Panel A, a domain in the COOH-terminal 99 amino acids of MxA (p10) forms a stable complex with sequences in the NH-terminal part of MxA (p60) to yield a GTPase-active molecule. Panel B, after treatment with proteinase K, nonconserved regions (which presumably serve spacer functions) are removed, resulting in a stable complex of p60 and p10 which retains GTPase activity. Panel C, GTPase activity is retained in deletion mutant His-MxA(301-576), which has most of the COOH-terminal p10 domain left intact. Panel D, complex formation is impaired in the COOH-terminal mutants of MxA which lack GTPase activity.



The results of our mutation analysis agree with the proposed structural model for MxA (Fig. 7) and suggest that back-folding of p10 is indeed required for GTPase activity of Mx proteins. Mutants of MxA which lack some COOH-terminal amino acids of p10 or which lack p10 altogether showed greatly reduced GTPase activity. By contrast, mutant His-MxA(301-576), which has a nearly unchanged p10 domain but a grossly truncated p60 domain, retained a high degree of GTPase activity. The proposed model can also account for our earlier observa-tion() that GTPase activity is rapidly and irreversibly lost upon treating MxA with denaturing agents. It is conceivable that such treatment opens up the p60-p10 complex and that re-formation of the complex may not occur spontaneously. We further observed that high yields of protease-resistant p60-p10 complex were only obtained with fresh MxA preparations that exhibited high specific GTPase activity. MxA preparations of low specific GTPase activity were degraded rapidly to small peptides, suggesting that the entire p60-p10 complex rather than the two individual polypeptides forms a compact tertiary structure. It will be of interest to crystallize the p60-p10 complex and to determine its structure.


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-79008Freiburg, 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. J. Virol. 69, in press.

The abbreviations used are: His-MxA, histidine-tagged MxA; PAGE, polyacrylamide gel electrophoresis; AMP-PNP, adenylyl-5`-,-imidotriphosphate; mant, N- methylanthraniloyl; GMP-PNP, guanylyl-5`-,-imidotriphosphate.

M. Schwemmle, F. Pitossi, and M. F. Richter, unpublished results.


ACKNOWLEDGEMENTS

-We thank Alfred Wittinghofer for support and helpful discussions and Annette Schwarz for expert technical assistance.


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