From the
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
The interferon-induced human MxA protein is a GTPase of 76-kDa
molecular mass which exhibits antiviral
activity
(1, 2, 3, 4, 6) .
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
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
Since the various MxA deletion mutants
contained the tripartite GTP-binding consensus element, which is
located in the NH
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
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
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
-We thank Alfred Wittinghofer for support and
helpful discussions and Annette Schwarz for expert technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)
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.
-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.
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.
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.
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.
-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).
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) .
-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.
(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.
,
-imidotriphosphate; mant, N-
methylanthraniloyl; GMP-PNP, guanylyl-5`-
,
-imidotriphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.