From the
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
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
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,
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).
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
To investigate further the substrate specificity of
His-MxA, we performed competition experiments with ATP, GDP, and GTP
analogs (GTP
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
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
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
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
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 MxA
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
We thank Jovan Pavlovic for numerous helpful
discussions at an early stage of this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
. 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.
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.
(
)
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) .
(
)
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.
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 GTP
S 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.
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).
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.
S 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,
GTP
S 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-MxA
mant-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).
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 GTP
S, 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 GTP
S,
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.
, 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.
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-MxA
mant-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-MxA
mant-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) .
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) .
GDP 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
values were calculated.
,
-imidodiphosphate; HPLC, high pressure liquid
chromatography; GMP-PNP, 5`-guanylyl-
,
-imidodiphosphate;
mant, N-methylanthraniloyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.