From the
Institute for Genetics, University of
Cologne, Zülpicher Strasse 47, 50674 Cologne, Germany and the
Department for Structural Biology, Max-Planck
Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund,
Germany
Received for publication, November 25, 2002 , and in revised form, April 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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Presently, with the possible exception of MxA (2), no explanation is available to connect the biochemical activity of these GTPases with their adaptive function in pathogen resistance. A member of the p65 GTPases from human cells, hGBP1, has been shown to block viral replication and endothelial cell proliferation (4, 10) and has been subjected to a detailed biochemical and structural analysis (1113). hGBP1 binds to GTP, GDP and GMP with equal affinity and hydrolyzes GTP to GDP and GMP. The GTPase activity of hGBP1 is stimulated at higher protein concentrations, and the presence of GTP or Gpp(NH)p favors the formation of oligomers. The nucleotide-free and the Gpp(NH)p-bound structures reveal many similarities and differences from other GTP-binding proteins. The high intrinsic GTPase activity, the cooperative hydrolytic behavior, and structural properties have suggested that hGBP1, representing the p65 GTPases, may be a functional and possibly also structural model for the family of large GTPases including Mx and dynamin (2, 14). In addition to cooperative GTPase activity, all of these GTPase families share the property of self-assembly into oligomers and higher order multimers, sometimes on specific templates such as lipid membranes in the case of dynamins (15, 16) and viral ribonucleoprotein particles in the case of MxA (2). The binding of GTP to these structures causes conformational changes that have been correlated with function and favors further self-assembly under some conditions (15, 1719). A critical and still unresolved issue with respect to dynamins is whether they function as conventional regulatory GTPases or as mechanochemical enzymes (20). In view of the proposed structural relationship between the protein families, the resolution of this issue will probably have implications not only for the function of dynamin but also for the mode of action of its interferon-inducible relatives, Mx and the p65 GBPs. The data that follow now add the p47 GTPases to this list.
IIGP1, with which this study is concerned, is a member of the p47
-interferon-inducible GTPase family. The mouse p47 GTPases so far
described form a distinct family with no sequence homology to other GTPases
outside the GTP-binding region
(3). The p47 GTPases have all
three GTP-binding motifs GXXXXGKS (P-loop), DXXG, and
(N/T)KXD clearly marked, unlike the p65 GTPase, hGBP1, where the base
specificity-related (N/T)KXD motif is functionally replaced by a
different structure (11). Four
members of the p47 GTPase family have been shown to mediate specific
resistance to intracellular pathogens. Fibroblasts stably expressing the p47
GTPase TGTP have reduced susceptibility to cytopathic effects of vesicular
stomatitis virus but not of herpes simplex virus
(5). Mice with targeted
disruptions of other p47 GTPases like IGTP, IRG-47, or LRG-47 are all
strikingly susceptible to Toxoplasma gondii infection; in addition,
the last also shows susceptibility to Listeria monocytogenes
infection (6,
7,
9).
In anticipation of a structural analysis of IIGP1, we here present a quantitative analysis of nucleotide binding and hydrolysis by the purified recombinant protein. IIGP1 is stable in the absence of nucleotides and has a relatively low affinity for guanine nucleotides in the micromolar range. The protein has higher affinity for GDP than for GTP, reflecting a higher dissociation rate for GTP. IIGP1 has low basal GTPase activity that is elevated at higher concentrations of the protein, suggesting cooperative behavior. When nucleotide-free or in the presence of GDP, IIGP1 behaves as a monomer, but it oligomerizes in the presence of GTP or with the nonhydrolyzable GTP analogue Gpp(NH)p. In summary, these properties place IIGP1 in the same functional category as Mx and GBP, the other large interferon-inducible GTPases. However, the distinctive properties of IIGP1 could serve as a key to understand the mechanism of action of this novel GTPase and its function in cell-autonomous pathogen resistance.
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EXPERIMENTAL PROCEDURES |
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Three versions of the IIGP1 protein are described in this paper, which differ at the C terminus, as shown in Table I. All three proteins were prepared and released from the glutathione affinity matrix by digestion of the GST fusion with thrombin as described above and carry the extension GSPGIPGSTT at the N terminus. The IIGP1-m protein originated from a cloning artifact; it differs from the wild-type by a 2-residue C-terminal truncation followed by the addition of 11 extra C-terminal residues. IIGP1-his has 6 histidine residues originally introduced as a C-terminal epitope tag.
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Nucleotide Binding Parameters
Three independent approaches were taken to measure the nucleotide binding
properties of IIGP1. Two of these relied on the use of
2',3'-O-N-methylanthraniloyl (mant)-modified
nucleotides. Mant nucleotides were used as fluorescent probes, since they
exhibit protein-binding properties similar to those of natural nucleotides.
The increase in mant fluorescence upon binding of the nucleotides to proteins
has been extensively used for the study of nucleotide binding interactions
with GTPases. The mant nucleotides used in our study (mGDP, mGTP, and the
nonhydrolyzable analogues mGTPS and mGpp(NH)p) were synthesized and
subsequently purified by ion exchange chromatography as described in Ref.
22. The purity of the
nucleotides was analyzed by reverse-phase chromatography. The concentration of
the nucleotides was determined by using molar absorption coefficients as
described above. Aluminum fluoride solutions were prepared by the addition of
300 µM aluminum chloride and 10 mM sodium fluoride.
The formation of AlF3 and
complexes is a pH-dependent
process, therefore denoted here as AlFx
(23). All experiments were
done with buffer 2 at 20 °C unless specified.
Stopped FlowIn stopped-flow experiments, at least a 5:1 ratio of protein to nucleotide concentrations were mixed together, providing conditions for pseudo-first-order binding kinetics. The time course of the increase in mant fluorescence upon binding of the protein to the mant nucleotides was recorded (SM-17; Applied Photophysics). Mant fluorescence was excited at 360 nm and monitored through a 405-nm cut-off filter. In each case, a single exponential function could be fitted to the data, yielding the observed rate constant kobs. A linear fit of the plot of kobs versus protein concentration was obtained, the slope of the curve denoting the association rate constant (kon) and the intercept showing the dissociation rate constant (koff). The Kd values are calculated from the ratio of koff and kon.
Equilibrium MeasurementsIIGP1 protein was titrated against mant nucleotides. The mant nucleotides (0.5 µM) were excited at 360 nm, and the fluorescence was monitored at 430 nm (Fluoromax 2; Spex Industries). The increase in fluorescence upon the stepwise addition of the protein was measured, and at each step the values were averaged over 1 min. The equilibrium dissociation constants, Kd, were obtained by fitting a quadratic function to the data as described in Ref. 24.
Isothermal Titration CalorimetryIsothermal titration calorimetry (Microcal Inc.) was used to measure the binding of nonlabeled nucleotides to IIGP1. The heat of binding was detected upon the stepwise addition of nucleotide into a cell containing the protein. The data were fitted using the manufacturer's software, yielding the enthalpy of the reaction, the dissociation constant (Kd), and the stoichiometry factor (N) as described in Ref. 25.
GTP Hydrolysis Assay
Different concentrations of GTP and protein were mixed and incubated at 37
°C in buffer 2. At different time points, aliquots were removed and
subjected to reverse-phase HPLC analysis for GTP and GDP. Samples were run
under isocratic conditions on a C18 column (0.4 x 25 cm
filled with 5-µm ODS Hypersil; Bischoff, Leonberg, Germany) with a
C18 prefilter (10 x 4.6 mm with 5-µm ODS Hypersil) in 7.5%
acetonitrile in buffer 3 (10 mM tetrabutylammonium bromide, 100
mM K2HPO4/KH2PO4 (pH
6.5), 0.2 mM NaN3) at a flow rate of 2 ml/min.
Nucleotides were detected at 252 nm in a UV absorption detector (Beckman
System Gold 166). The concentration-dependent GTPase activity of the three
forms of IIGP1 protein (Table
I) was measured at a GTP concentration of 700 µM and
at a range of protein concentrations.
Dynamic Light Scattering
Dynamic light scattering was performed using a DynaPro molecular sizing
instrument (Protein Solutions) equipped with a temperature control unit. The
sample was filtered through a microsampler syringe, where 50 µl of the
sample was passed through a filtering device using a 0.02-µm filter into a
12-µl Quartz cuvette. The sample (either protein alone or protein with
different nucleotides) in buffer 2 was illuminated by a 25-milliwatt, 750-nm
wavelength solid state laser. The time scale of scattered light intensity
fluctuation for each measurement was evaluated by autocorrelation, from which
the translational diffusion coefficient (DT) was
calculated. The Mr was estimated from the hydrodynamic
radius Rh, which was derived from DT
using the Stokes-Einstein equation and the sample temperature (T = 4 °C)
using the standard curve of Mr versus Rh for
globular proteins. The data was analyzed by Dynamics 4.0 and Dyna LS
software.
A qualitative measurement to show oligomerization was also done in a conventional fluorescence spectrometer, where the sample (50 µM IIGP1-wt and 1 mM nucleotide) was excited at 350 nm and monitored at the same wavelength.
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RESULTS |
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Nucleotide Binding
Equilibrium TitrationsThe binding parameters of IIGP1-m for
guanine nucleotides were determined at equilibrium in titrations of protein
against constant mant nucleotides. Fig.
2a shows the titration curve for the increase in
fluorescence upon the binding of mant-GDP (mGDP) to IIGP1-m. IIGP1-m was added
from a 600 µM stock solution, and at each step the values for
the increase in fluorescence were averaged over a period of 1 min.
Dissociation constants were calculated from curves fitted to the data
following Ref. 24. The
Kd value for nucleotide binding is in the
micromolar range, and mGDP shows higher affinity than mGTPS
(Table II). mGpp(NH)p, another
nonhydrolyzable analogue of GTP, shows weak binding in comparison with the
other nucleotides (Kd of
130
µM). Interestingly, we could not detect any increase in
fluorescence upon the addition of aluminum fluoride (AlFx)
to IIGP1.mGDP solution (data not shown), and in confirmation, no decrease in
the dissociation rate of mGDP could be detected by stopped flow in the
presence of AlFx. Thus, monomeric IIGP1 in free solution
in the presence of GDP appears not to be able to stabilize the transition
state of GTP hydrolysis through binding of AlFx.
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The interactions of unlabeled GDP and GTPS with IIGP1-m were
additionally measured by isothermal titration calorimetry in order to
investigate possible interference of the mant group in the fluorescence-based
binding assay. Fig. 2b
(upper lane) shows the raw data for binding upon the stepwise
addition of GDP to IIGP1-m. The exothermic process is reflected in a negative
power pulse from which the heat of the reaction is calculated by integration.
The enthalpy of the reaction normalized to the concentration of injected GDP
was plotted against the molar ratio of GDP and IIGP1-m as shown in the lower
panel. The parameters defining the theoretical curve yielded a
Kd for this interaction of 2.5 µM
and an enthalpy of association of 15 kcal/mol. In addition, 1:1
stoichiometry is evident from the data. The results from
Table II suggest that the mant
modification does not significantly influence the binding affinity of GDP and
GTP
S for IIGP1.
Dynamics of Nucleotide BindingThe dynamics of mant
nucleotide binding to IIGP1 were determined by stopped flow. mGTPS was
used to control for potential complications in the measurement of GTP binding
caused by hydrolysis. No significant difference was seen (Tables
II and
III), no doubt due to the slow
turnover of GTP by IIGP1 (see below). The IIGP1 protein was used in a large
molar excess over the nucleotide in order to obtain pseudo-first-order
reaction kinetics (shown for IIGP1-m in
Fig. 3). A single exponential
function was fitted to the data, where the resulting time constant corresponds
to the inverse observed rate constant kobs
(Fig. 3, upper inset).
The observed rate constants for the binding of mGDP were plotted against
IIGP1-m concentration. The slope of the straight line denotes the association
rate constant (kon), and the intercept shows the
dissociation rate constant (koff).
koff was also measured in a displacement experiment by the
addition of excess unlabeled nucleotide to IIGP1-mGDP preformed complex. This
leads to a quasi-irreversible dissociation of mGDP and therefore to a single
exponential decrease of fluorescence as shown in the lower inset in
Fig. 3.
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The Kd values of different nucleotides to
IIGP1-m using equilibrium methods are compared with
Kd from stopped-flow experiments in
Table II. The three different
methods gave comparable results. The nucleotide binding affinities determined
by stopped flow for the native IIGP1 protein and for the two forms varying at
the C terminus (Table I) are
shown in Table III. It is
apparent that the Kd values and the rate
constants are similar for all three proteins, suggesting that the C terminus
is not involved in nucleotide interaction with IIGP1 (see below). The
association rate constants are similar for the different nucleotides, but the
dissociation rate constants vary significantly. The smaller
koff for mGDP accounts for the higher affinity compared
with mGTP and mGTPS, as observed in the equilibrium titrations
described above (Table II). To
summarize the results from three different methods and from the three
C-terminal versions of IIGP1, the dissociation constant for GDP is
1
µM, whereas for GTP it is
15 µM.
IIGP1 as a GTPase
In all GTPases, the identity of the nucleotide bound to the protein and the
rate of conversion of GTP to GDP by hydrolysis are crucial factors determining
their functional regulation
(26). We therefore document a
detailed analysis of the kinetics of GTP hydrolysis by IIGP1. The rate of
hydrolysis and product formation was analyzed by reverse-phase HPLC. The
activity of the three forms of IIGP1 (IIGP1-wt, IIGP1-m, and IIGP1-his) at
2100 µM was analyzed over GTP concentrations from 0.05 to
2 mM. ATP was not hydrolyzed, confirming nucleotide specificity and
indicating that our preparations were free of phosphatases (data not
shown).
IIGP1 hydrolyzes GTP to GDP and not to GMP. A representative curve for the time course of GTP hydrolysis by IIGP1-wt is shown in Fig. 4a. The complete hydrolysis of 700 µM GTP to GDP with 50 µM IIGP1 at 37 °C is documented. The rate constant for GTP hydrolysis was obtained from the initial rates, since later values are affected by product inhibition. Magnesium ions were found to be essential for the GTPase reaction; severely impaired GTPase activity was observed in the presence of 10 mM EDTA (Fig. 4b). Surprisingly, in view of the results reported above, where GDP binding to IIGP1 could not be directly stabilized by AlFx, the hydrolysis of GTP by IIGP1 was greatly inhibited by the addition of AlFx, suggesting that there are conditions under which AlFx can stabilize the GDP-bound state and inhibit nucleotide exchange (Fig. 4b). The catalytic activity of IIGP1 at different protein and nucleotide concentrations was determined, as shown in Fig. 4c. The Michaelis-Menten equation was applied to a plot of specific activity (at a range of protein concentration) versus substrate concentration. Intriguingly, a small increase in Km was observed with increased protein concentration, suggesting functional cooperativity between IIGP1 molecules.
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In order to investigate the cooperativity effect more precisely, the dependence of the specific GTPase activity on IIGP1 concentration was measured at a fixed GTP concentration of 700 µM as shown in Fig. 4d (closed circles, IIGP1-m; open circles, IIGP1-wt). The maximum specific rate of GTP hydrolysis by IIGP1 was close to 2 min1 (see Fig. 4c, where 50 µM IIGP1-m and 2 mM GTP was used). The increase in activity of the protein with an increase in protein concentration observed in the case of IIGP1-wt and IIGP1-m demonstrates a cooperative mechanism of GTP hydrolysis. Intriguingly, the GTP hydrolysis rate of IIGP1-his C-terminally modified protein showed marginal if any protein concentration dependence over the tested range, remaining at or close to the basal rate of 0.1 min1 (Fig. 4d, closed triangles).
GTP-dependent Formation of IIGP1 Oligomers
Other GTPases that show cooperative activity have also been shown to form
enzymatically active oligomers in the presence of nucleotides (reviewed in
Ref. 2). The oligomerization of
IIGP1-m was investigated using dynamic light scattering at 4 °C at protein
concentrations of 50100 µM and nucleotide (GDP, GTP, and
Gpp(NH)p) concentrations of 1 mM. The change in the Rh of
IIGP1 (derived from the unimodal fit analysis by Dynamics 4.0 software)
versus time was plotted for each of the data sets with and without
nucleotides. Fig. 5a
shows the time-dependent oligomerization of IIGP1-wt with GTP and Gpp(NH)p. A
striking increase in hydrodynamic radius was detectable after the addition of
GTP and also, but to a lesser degree, after the addition of Gpp(NH)p. No
detectable higher molecular forms were found with the apoprotein
(nucleotide-free) or in the presence of GDP, since the Rh is 3.1 nm,
which corresponds to the expected molecular mass of 47 kDa. The difference in
oligomer formation between GTP- and Gpp(NH)p-bound forms could be due to their
difference in nucleotide binding affinities.
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As shown in Fig. 4d, IIGP1 modified by the addition of a C-terminal His6 tag showed essentially no cooperative GTPase activity. It was therefore of interest to know whether the C-terminal modification also blocked the formation of nucleotide triphosphate binding oligomers. Using the dynamic light scattering assay, IIGP1-his showed no oligomerization in the presence of GTP and Gpp(NH)p (Fig. 5a) where the curve for IIGP1-his in the presence of GTP overlays with nucleotide-free IIGP1-wt, IIGP1-wt GDP. The hydrodynamic radius of IIGP1-his in the presence of GTP persists at around 3 nm, thus a monomer.
Since oligomerization was possible only in the presence of GTP or Gpp(NH)p, and not in the presence of GDP, the reversibility of these oligomers was tested with respect to GTP hydrolysis. In order to show directly the relationship between IIGP1-catalyzed GTP hydrolysis and oligomer formation, we used light scattering in a fluorescence cuvette from which aliquots for nucleotide analysis were taken simultaneously. The time course of the oligomerization in a solution of GTP (1 mM) and IIGP1-wt (50 µM) is shown in Fig. 5b. The initial rise in light scattering was followed by a slow decrease associated with progressive hydrolysis of GTP. This time-dependent oligomerization led to visible turbidity, which later reverted to a clear solution. The decrease in turbidity is therefore not due to flocculation (sedimentation in the cuvette) but presumably due to GTP hydrolysis and dissolution of the oligomers. The accumulation of GDP inhibits GTP-dependent oligomerization efficiently through the relatively high binding affinity of GDP for IIGP1. The light scattering trace in Fig. 5b showing the reversibility of the IIGP1 oligomers is plotted along with the time course of GTP hydrolysis (for the same nucleotide and protein concentrations) to delineate the relationship between the two processes. The reversibility of the oligomers was not seen in the previous experiment using dynamic light scattering due to the short time ranges of the measurements. With this experimental system, we also wanted to analyze the binding of AlFx to IIGP1. As shown in Fig. 5c, the high light scattering of IIGP1 solution following the addition of GTP does not decay in the presence of AlFx, consistent with the observed inhibition of GTP hydrolysis by AlFx (Fig. 4b).
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DISCUSSION |
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Due to slow dissociation rates, Ras and EF-Tu bind GDP very tightly
(picomolar and nanomolar Kd values), and GDP
release is accelerated by the exchange factors Sos and EF-Ts, respectively
(30,
36,
37). For IIGP1, the nucleotide
dissociation rate constants are large, 21 s1 for
GTP and 3.6 s1 for GDP. This indicates very short
lifetimes for the complexes as compared with Ras-like GTPases, G or
EF-Tu. Given the cellular concentrations of GTP and GDP
(38), the molar ratio of the
GDP and GTP states of IIGP1 should be roughly 3:1 in favor of GDP, but
switching states might well not require a putative guanine nucleotide exchange
factor because of the high dissociation rates. In contrast to these
expectations derived from consideration of IIGP1, a direct investigation of
bound nucleotide from the ex vivo purified p47 GTPase, IGTP, for
which unfortunately no quantitative nucleotide binding parameters are
available, showed only detectable bound GTP
(39). It may be, however, that
the ratio of GTP-bound and GDP-bound forms is altered in vivo by
interaction partners that change the relative affinities for nucleotides.
Until now, no interaction partners have been described for any of the p47
GTPases.
GTPase activity has been shown to be crucial for the regulation of almost
all GTPases. With respect to the switch GTPases, the time from the binding of
GTP to its hydrolysis is critical for downstream cellular functions and is
therefore targeted for precise modulation. For example, Ras has a very low
intrinsic hydrolytic rate (0.03 min1). Here,
functional efficiency is achieved by accelerating the turnover rate by a
factor of 105 by an activating protein, RasGAP
(40). In contrast, the class
of large GTPases exemplified by dynamins shows a mechanism in which increased
GTPase activity is associated with self-assembly. The homo-oligomerization of
dynamins has been shown to be essential for its biological function and is
nucleotide-dependent (15,
41,
42). The homologous Mx
proteins, which, like the p47 GTPases, are induced by interferons and function
in pathogen resistance, also show increase in GTPase activity through
nucleotide-stimulated self-association
(2,
18,
19). There is evidence that
self-association of Mx may play a role in its viral resistance function.
Similarly, the p65 interferon-induced GTPase hGBP1 also shows
concentration-dependent GTPase activity and nucleotide-dependent
oligomerization (11,
13), although in this case
there is no evidence yet that these properties play an in vivo role.
The dynamins, Mx proteins, and the GBPs, despite showing limited (dynamin
versus Mx) or no (GBP proteins versus Mx or dynamins)
sequence homology between families, appear thus to share some distinctive
properties. Some additional structural similarities have been predicted, like
an assembly domain and an intrinsic C-terminal GAP domain regulating their
function through interaction with the N-terminal G-domain
(14). Our data show that IIGP1
exhibits several biochemical properties that recall the dynamins, Mx proteins,
and hGBP1 protein, namely relatively low affinity for guanine nucleotides
(micromolar Kd values), cooperative GTPase
activity, and nucleotide-dependent oligomerization. However, IIGP1 clearly
differs from them in its higher affinity for GDP than for GTP, high GTP
dissociation rate, and low GTPase activity (2
min1 under saturating conditions). IIGP1 GTPase
activity may rather be similar to Ras-like GTPases or G, where a
further activating interaction (in these cases with RasGAP and RGS) is
required to accelerate the rate of GTP hydrolysis. In the case of IIGP1,
association of the protein with intracellular membranes may contribute to a
further acceleration of GTPase activity, as observed for dynamins
(16). The cooperative activity
and GTP-dependent oligomerization of IIGP1 are likely to be essential for
regulating its function in vivo, although there is no direct evidence
for IIGP1 forming stable oligomers under in vivo conditions.
Increased GTPase activity upon oligomerization has also been described for the
small GTPase Rac1, and a functional significance for the activation of the
dimeric effector Pak has been documented
(43). In this case, however,
the formation of oligomers is independent of the presence of nucleotide, a
property that distinguishes Rac1 from the large self-associating GTPases.
The IIGP1-his protein modified at the C terminus shows neither GTP-dependent oligomerization nor concentration-dependent GTPase activity, thus laying emphasis on the significance of the C terminus in the formation of oligomers. We do not see any influence of the histidine tag in IIGP1-his on nucleotide binding, suggesting that the tag interferes with oligomerization rather than shielding or delaying the GTP binding/hydrolysis. The properties of IIGP1-his strengthen the argument presented here that oligomerization plays a role in acceleration of GTPase activity. The other C-terminal modified form of IIGP1 studied here, IIGP1-m, appears to be essentially wild-type despite the C-terminal extension.
AlFx essentially plays the role of the leaving
-phosphate and mimics the transition state of GTP hydrolysis
(44). For small GTPases in the
GDP-bound state, AlFx binding has been shown only in the
presence of a GAP, which introduces the stabilizing arginine residue (termed
the "arginine finger"). In contrast, the G
subunit is
capable of binding GDP.AlFx, since it harbors an internal
GAP domain providing the arginine finger. Recently, AlFx
binding to hGBP1-GDP in the absence of an external GAP has been documented
(11). In the interests of
consistency, this result can be explained by the proposition that hGBP1 might
have an internal GAP domain like the G
. Here, we were not able to
demonstrate binding of AlFx to IIGP1.GDP by mixing these
two complexes. Nevertheless, binding of AlFx to IIGP1 is
indicated in the oligomeric state that is formed when GTP is bound and being
hydrolyzed. Our interpretation of these observations is that
AlFx binds to IIGP1.GDP when the oligomer is preformed,
representing the catalytically active state. A plausible conclusion from these
results within the Ras and G
paradigm would be that an
additional catalytic residue might be provided in trans by an
adjacent monomer, as proposed for dynamins in the oligomeric state.
The catalytic residues like Gln61 in Ras or the arginine finger cannot be identified in the family of large GTPases. IIGP1 has an Ile in the place of Ras Gln61; indeed, all members of the family of large GTPases, the dynamins, Mx, and hGBP1, have a hydrophobic residue as in IIGP1, suggesting that the whole group employs a different catalytic mechanism from that of the small GTPases. The unusual character of the p47 GTPase reaction mechanism is further emphasized by the presence of a highly anomalous methionine in place of lysine at the canonical GK(S/T) sequence of the P loop motif in three members of the family, IGTP, LRG-47, and GTPI (3). Despite this substitution, which is unique to this subfamily of p47 GTPases (45), IGTP has been shown to have GTPase activity (46). Detailed structure/function studies will be required to illuminate the reaction mechanism of the p47 GTPases.
IIGP1 is localized in the membrane of the endoplasmic reticulum and Golgi (47), and membrane localization is at least partly dependent on an N-terminal myristoyl modification.2 Both dynamins and Mx proteins interact with lipid membranes, which become deformed by the formation of structured oligomers (2, 17). hGBP1 carries a canonical C-terminal CaaX motif, predicting isoprenylation and membrane association (48). The interaction of all of the large self-associating GTPase families with membranes may imply that the interferon-inducible members of these groups exercise their antipathogenic function through modification of aspects of membrane structure or behavior that are normally exploited by intracellular pathogens.
A deeper understanding of the mechanism by which p47 GTPases interfere with
intracellular pathogens demands an investigation not just into the nucleotide
binding properties and enzyme kinetics of the purified proteins but rather a
detailed understanding of their properties and regulation in vivo. It
is likely that association with other proteins exercises regulatory control
over IIGP1 in vivo, whereas the precedent of ADP-ribosylation
factor-1 may suggest that the association with intracellular membranes may
also be highly regulated, perhaps via the myristoyl group, and itself of
importance in determining the in vivo properties of the protein
(49,
50). Identification of the
protein neighbors or regulators of IIGP1 in its native environment in the
interferon--induced cell will help us further in analyzing the
significance of oligomerization and regulated GTPase activity as well as
membrane association for the function of the protein in vivo.
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FOOTNOTES |
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** Supported by a grant from the Boehringer Ingelheim Fonds.
¶ To whom correspondence may be addressed. Tel.: 49-221-470-4864; Fax: 49-221-470-5015; E-mail: jonathan.howard{at}uni-koeln.de.
|| To whom correspondence may be addressed. Tel. 49-231-133-2160; Fax: 49-231-133-2199; E-mail: christian.herrmann{at}mpi-dortmund.mpg.de.
1 The abbreviations used are: GBP, guanylate-binding protein; mant,
2',3'-O-N-methylanthraniloyl; GTPS,
guanosine 5'-3-O-(thio)triphosphate; mGTP
S,
mant-GTP
S; Gpp(NH)p, guanosine
5'-(
,
-imido)-triphosphate; mGpp(NH)p, mant-Gpp(NH)p; HPLC,
high pressure liquid chromatography; mGDP, mant-GDP; GST, glutathione
S-transferase.
2 R. C. Uthaiah and J. C. Howard, unpublished results.
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REFERENCES |
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