The rate of hydrolysis of GTPcNH2bound to Ras protein lay between the rates found for Ras-bound GTPcS and GppNHp, while Ras-catalysed hydrolysis of GTPcF was almost as fast as for GTP..
Trang 1Synthesis, characterization and application of two nucleoside
Michael Stumber1, Christian Herrmann2, Sabine Wohlgemuth2, Hans Robert Kalbitzer1, Werner Jahn1 and Matthias Geyer1,*
1
Max-Planck-Institut fu¨r medizinische Forschung, Department of Biophysics, 69120 Heidelberg, Germany;
2
Max-Planck-Institut fu¨r molekulare Physiologie, Department of Structural Biology, 44227 Dortmund, Germany
Guanosine triphosphate nucleotide analogues such as
GppNHp (also named GMPPNP) or GTPcS are widely
used to stabilize rapidly hydrolyzing protein-nucleotide
complexes and to investigate biochemical reaction
path-ways Here we describe the chemical synthesis of guanosine
5¢-O-(c-amidotriphosphate) (GTPcNH2) and a new
synthe-sis of guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF) The
two nucleotides were characterized using NMR
spectrosco-py and isothermal titration calorimetry Chemical shift data
on 31P,19F and1H NMR resonances are tabulated For
GTPcNH2 the enthalpy of magnesium coordination is
DH ¼ 3.9 kcalÆmol)1 and the association constant Ka is
0.82 mM )1 The activation energy for GTPcNH2ÆMg2+
complex formation is DH¼ 7.8 ± 0.15 kcalÆmol)1, similar
to that for the natural substrate GTP For GTPcF we ob-tained a similar enthalpy of DH ¼ 3.9 kcalÆmol)1while the magnesium association constant is only Ka¼ 0.2 mM )1 The application of both guanine nucleotide analogues to the GTP-binding protein Ras was investigated The rate of hydrolysis of GTPcNH2bound to Ras protein lay between the rates found for Ras-bound GTPcS and GppNHp, while Ras-catalysed hydrolysis of GTPcF was almost as fast as for GTP The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies
Keywords: nucleotides; nucleotide analogues; NMR spectro-scopy; GTP hydrolysis; Ras
Nucleotides are fundamental components in cellular
meta-bolism Acting as substrates for nucleotide binding proteins,
they are the protagonists of a large variety of cellular
processes Nucleotides can regulate enzymatic activity by
transitions between their mono-, di- and triphosphate
bound forms These transitions often induce conformational
changes in the proteins, referred to as the active and
inactive conformations Perhaps the best known example is
the energy metabolism of adenosine nucleotides: hydrolysis
of ATP to ADP leads to functional molecular
rearrange-ments in the actomyosin mediated muscle contraction
Guanine nucleotide-binding proteins on the other hand are
specialized in the control of intracellular communication
processes such as signal transduction (Ras and Rho families) or protein and vesicle trafficking (Ran and Rab families, respectively), which are combined with GTP-hydrolysis (reviewed in [1–3]) Another aspect of nucleotide mediated transformation is the transfer of the leaving phosphoryl group (mostly the c-phosphate group) to acceptors like water, amino-acid residues, or other nucleo-tides Often the association of a metal ion, usually magnes-ium, with the phosphate groups of the nucleotide is crucial for these events
The study of nucleotide-binding proteins, their function, structure and mechanism, often demands use of nonhy-drolyzable or slowly hynonhy-drolyzable nucleotide analogues These modifications become necessary when stabilization of
a specific isoform of the protein is required In cellular assays the triphosphate analogues GTPcS and ATPcS are most commonly used, usually in order to generate the constitutively active form of a protein In structural biology, long-term stability of the protein-nucleotide complex is required in order to grow homogeneous crystals or to obtain
a single state of the protein Here, the most commonly used triphosphate analogues are GppNHp (also named GMPPNP or GDPNP) and to a minor extent GppCH2p (also named GMPPCP) and their respective adenosine counterparts AppNHp and AppCH2p Another application
of substrate analogues is the use of caged nucleotides to characterize unstable protein intermediates by X-ray crys-tallography [4] Nucleotide modifications can also serve as
an approach to designing dominant negative forms of a protein [5] or to solve the phase problem in crystallography [6] Even more specific is the application of aluminium fluoride, beryllium fluoride or orthovanadate in the presence
Correspondence to M Geyer, Max-Planck-Institut fu¨r medizinische
Forschung, Abteilung Biophysik, Jahnstraße 29,
D-69120 Heidelberg, Germany.
Fax: + 49 6221 486 437, Tel.: + 49 6221 486 396,
E-mail: geyer@mpimf-heidelberg.mpg.de
Abbreviations: GTPcNH 2 , guanosine 5¢-O-(c-amidotriphosphate);
GTPcF, guanosine 5¢-O-(c-fluorotriphosphate); GppNHp, guanosine
5¢-O-(b,c-imidotriphosphate); GppCH 2 p, guanosine
5¢-O-(b,c-methylenetriphosphate); GTPcS, guanosine
5¢-O-(c-thiotriphos-phate); ITC, isothermal titration calorimetry; DCC,
dicyclohexylcar-bodiimide; DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate;
THC, triethylammonium hydrogencarbonate.
*Present address: Max-Planck-Institut fu¨r molekulare Physiologie,
Department of Physical Biochemistry, 44227 Dortmund, Germany.
(Received 23 January 2002, revised 8 May 2002,
accepted 17 May 2002)
Trang 2of a nucleoside diphosphate These compounds can form
stable analogues that mimic the transition state of the
terminal leaving group of the nucleotide within a
protein-nucleotide complex [7,8]
Mechanistic studies to analyse the enzymatic activity of a
nucleotide binding protein usually benefit from the
avail-ability of a broad range of different nucleotide phosphate
analogues Here, advantage can be taken of the individual
characteristics of the nucleotide when applied to a protein
Differences in metal ion binding properties as well as charge
distribution and hydrophobicity determine the specific
features of a nucleotide that provide insights into the
biological system Also, nucleotide modifications such as
spin labeling make the protein-nucleotide complex
access-ible to spectroscopic techniques Most prominent is the use
of fluorescent analogues (e.g mant-GTP) for kinetic
measurements by fluorescence spectroscopy and17
O-labe-ling for EPR or NMR techniques
Here we investigate two modified nucleoside
triphos-phates which are stable and show distinct characteristics:
guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH2), for
which we describe the first synthesis, and guanosine
5¢-O-(c-fluorotriphosphate) (GTPcF) [9], which we synthesized
by the method of Wittmann [10] Both are shown in Fig 1
We characterized the stability and metal ion binding properties of the two nucleotide analogues by NMR spectroscopy and isothermal titration calorimetry Both nucleotides were bound to the small GTP-binding protein Ras and the rates of hydrolysis were determined in comparision to other nucleotide triphosphate derivatives Finally, the suitability for spectroscopic and structural studies was tested by formation of the complex between RasGTPcNH2 and the Ras-binding domain of the Ras effector protein c-Raf-1
M A T E R I A L S A N D M E T H O D S
General description of synthesis High pressure liquid chromatography (HPLC) was done on
a Beckman System Gold Nucleotides were analysed by ion-pair chromatography on a reversed phase Super ODS column, 50· 4.6 mm (TOYOPEARL) at a flow rate of 1.2 mLÆmin)1, using a linear gradient from 100% 10 mM
tetrabutyl-ammonium bromide/10 mM sodium phosphate buffer (pH 6.8) to 100% acetonitrile within 10 min Detec-tion was at 260 and 340 nm The retenDetec-tion times given are for orientation only
GTP-triethylammonium salt was prepared by applying GTP sodium salt to a Super Q column (TOYOPEARL) and elution with a gradient from 0 to 1M triethylammo-nium hydrogencarbonate (THC) The eluate containing the nucleotide (retention time in HPLC 4.65 min) was evapor-ated under reduced pressure, redissolved in methanol, again evaporated and dried over P4O10 Monoamido-phosphoric acid, H2PO3NH2, was prepared as described [11]
Synthesis of GTPcNH2and GTPcF
To the solution of 0.8 g GTP triethylammonium salt in
5 mL dimethylsulfoxide were added 0.8 g DCC and 80 mg pyridinium hydrochloride After 20–24 h at room tempera-ture the mixtempera-ture was treated with about 5 mL concentrated ammonia in water for 30 min The solution was diluted with 60–70 mL water and, after filtration, applied to a Super Q column (2.5· 20 cm) The column was eluted at a rate of
5 mLÆmin)1 with a gradient from 0 to 1 M THC within
120 min Fractions containing the GTPcNH2(as checked
by UV absorption and HPLC, retention time 4.20 min) were collected and evaporated Any remaining THC was removed by dissolving in methanol and repeated evapor-ation under reduced pressure, yield of the pure GTPcNH2 was 50–60%
One gram of GTP triethylammonium salt was added to a stirred solution of 2.5 mL tributylamine and 1.2 g 2,4-dinitrofluorobenzene in about 10 mL dimethylforma-mide After 6–8 h a clear solution was obtained The mixture was kept for 20–24 h at room temperature The crude product was precipitated with 100 mL acetone and
300 mL diethyl ether The pellet was dissolved in water (about 20 mL) and applied to a Super Q column (18· 2.6 cm) The column was eluted with a gradient of 0–1M THC within 2 h at a flow rate of 5 mLÆmin)1 Fractions containing the reaction product (retention time of 4.47 min, no absorption at 340 nm) were collected and evaporated as described for the GTP triethylammonium salt The product was dissolved in 20 mL methanol and
Fig 1 Chemical structure of the nucleoside triphosphate analogues
synthesized (A) Guanosine 5¢-O-(c-amidotriphosphate) (GTPcNH 2 )
and (B) guanosine 5¢-O-(c-fluorotriphosphate) (GTPcF) Displayed is
the -riboside form of the respective nucleoside.
Trang 3precipitated by addition of a solution of 250 mg NaClO4in a
few ml methanol to remove part of the colored by-products
The pellet was dissolved in water (20 mL) and purified on a
Super Q column as described above, giving an almost
colorless substance (yield in the range 5–10%)
Preparation of NMR samples and NMR spectroscopy
31P and19F NMR spectra of free nucleotides were recorded
in aqueous solution of 90%/10% H2O/D2O Typically the
lyophilized nucleotide was redissolved to a final
concentra-tion of 2–10 mMand 2500 lL of the sample volume was
placed in 10 mm NMR tubes (Wilmad) For titration
experiments various amounts of MgCl2were added from a
100-mM stock solution Proton NMR experiments were
performed using 500 lL sample volume in 5 mm NMR
tubes (Wilmad).31P NMR spectra of C-terminal truncated
wildtype Ras protein (residues 1–167) complexed with
GTPcNH2ÆMg2+were recorded in 40 mMTris/HCl, 5 mM
MgCl2and 2 mMDTE at pH 7.4 Here, sample volumes of
2500 lL of 1.0 mM concentrated protein were measured
containing 10% D2O
1H,19F and31P NMR experiments were performed on a
Bruker AMX-500 NMR spectrometer working at
reson-ance frequencies of 500 MHz, 470 MHz and 202 MHz,
respectively.31P spectra were referenced to 85% phosphoric
acid enclosed in a glass sphere which was immersed in the
sample and calibrated for various temperatures.19F spectra
were referenced to trifluoroacetic acid, based on the IUPAC
conventions for indirect referencing relative to internal DSS
[12] Unless noted otherwise, phosphorus spectra were
recorded at 20C with a total spectral width of 60 p.p.m
For one dimensional31P NMR spectra of free nucleotides,
64–512 free induction decays were summed after excitation
with a 65 degree pulse using a repetition time of 3–5 s A
total of 32 K time domain data points were recorded and
transformed to 16 K real data points corresponding to a
digital resolution of 0.74 Hz point)1 The 31P spin-spin
coupling constants of the nucleotide-Mg2+complexes were
determined from a nonfiltered 1D spectrum with a digital
resolution of 0.25 Hz per point after Fourier
transforma-tion
All spectra were processed on a Silicon Graphics Indigo2
workstation using the software package UXNMR (Bruker,
Karlsruhe) for data processing and data evaluation
Phos-phorus spectra used for exchange rate determination were
filtered by an exponential window function causing no
significant line broadening
Determination of exchange rates
The Mg2+ exchange rates of GTPcNH2 were extracted
from a series of31P NMR exchange spectra The spectra
were analyzed and compared to simulations based on the
mathematical treatment of the exchanging spin system
following Nageswara Rao [13] The simulation of the31P
spectra was built on C++ NMR library GAMMA [14],
modeling a three spin system with an ABC fi A¢B¢C¢
exchange Chemical shift and J-couplings were determined
for NMR spectra of both pure states: without magnesium
complexation (state A) and with saturated magnesium
complexation (state B) (see Tables 1 and 2) Thus, the only
parameters to be adjusted were the relative populations of
the states A and B and the exchange rates k1for A fi B and k)1for B fi A As B ¼ 100%–A and k)1¼ k1*A/B only two free parameters had to be fitted to the experimental data The simulations were performed on the complete31P spectra (a-, b-and c-phosphorus nuclei), using chemical shift values and J-coupling constants as listed in Tables 1 and 2
Isothermal titration calorimetry The interaction between a nucleotide and the magnesium ion was investigated by means of ITC (ITC-MCS, Micro-Cal, Inc.) Briefly, in such an apparatus the solutions are thermostatted to the desired temperature, the nucleotide at 5.0 mM placed in a cell which is accurately temperature controlled and the MgCl2solution at 50 mM in a syringe dipping into the cell The two solutions are mixed by computer controlled stepwise injections (typically in inter-vals of 4 min) from the syringe which serves at the same time as a stirrer The heat consumed due the endothermic association process is measured by the detection of the heating power which is necessary to keep the cell at constant temperature [15] All ITC experiments were performed at
25C The data were analyzed using the manufacturer’s software yielding the stoichiometry N, the binary equili-brium association constant Ka¼ [nucleotideÆMg2+]/ [nucleotide]/[Mg2+] and the enthalpy of association DH, the latter with the approximation that this parameter is independent of the concentration The change of entropy DS is calculated by the fundamental relationship –RT lnK ¼ DH – TDS The experimental error on DH
Table 1 NMR chemical shifts of GTPcNH 2 , G TPcF, and GTP in aqueous solution Spectra were recorded in 90%/10% H 2 O/D 2 O at
pH 7.4 and 25 C 31 P and 19 F chemical shifts were referenced to 85% phosphoric acid and trifluoroacetic acid, respectively, using the indirect reference method with parameters adopted from IUPAC [12].
31 P chemical shift d (p.p.m.)
19 F chem.
GTPcNH 2 ÆMg 2+ )11.33 )21.50 )0.34 –
GTPcÆFMg 2+
)11.89 )23.19 )18.63 0.97
Table 2 J-Coupling constants of GTPcNH 2 and GTPcF in aqueous solution.
J-coupling constants (Hz) Nucleotide 2J PaPb
2
J PbPc
1
J PcF
Trang 4is 5% whereas the experimental error on Kais about 10–
20% In addition, the stoichiometry factor N is obtained
from the fit to the data, where a value of 1 corresponds to
1 : 1 complex formation
Protein preparation and guanine nucleotide exchange
In order to test the applicability of the two synthesized
triphosphate nucleotide analogues to nucleotide binding
proteins, the small GTP-binding protein Ras (residues
1–167) was synthesized in Escherichia coli and purified as
described [16] Purified GDP, GTP, GTPcS and GppNHp
reagents were purchased from Sigma and GppCH2p was
ordered from JenaBioScience GDP, which binds very
tightly to Ras, was replaced with the respective GTP
analogue by the following procedures For nucleotide
exchange GTPcNH2, GppNHp and GppCH2p were each
incubated at threefold molar excess with Ras in the presence
of 200 lMammonium sulfate, 0.1 lMzinc chloride and 1 U
alkaline phosphatase per mg Ras overnight at 4C In order
to load Ras with GTP, GTPcF, or GTPcS nucleotide-free
Ras was produced by incubation overnight at 4C in the
presence of 200 lMammonium sulfate, 0.1 lMzinc chloride
and 0.2 U alkaline phosphatase per mg Ras After size
exclusion chromatography, one of the nucleotides was then
added to the Ras protein Excess nucleotide after either
procedure was removed (which is important in order to
obtain accurate single turnover hydrolysis rate constants)
The pooled Ras fractions were concentrated to 20 mgÆmL)1
by centrifugal concentrators (Vivaspin 10 kDa cut-off,
VivaScience) The buffer used in all these procedures
contained 25 mMTris/HCl at pH 7.4, 2.5 mMMgCl2, and
1 mM DTE The Ras catalysed nucleotide hydrolysis was
determined with HPLC by measuring the concentration of
protein-bound GTP or its triphosphate analogues and GDP
as described [17] Intrinsic reaction rates were obtained
from the decay of the (triphosphate nucleotide)/(tri-and
diphosphate nucleotide) ratio with time, fitted to
single-exponential curves The Ras-binding domain of human
c-Raf-1 (Raf-RBD, 81 residues) was expressed in E coli
and purified as described recently [18]
R E S U L T S
NMR spectra, chemical shift data and J-coupling
constants of the two nucleotides
Proton, phosphorus and fluorine NMR measurements
confirmed the chemical structure and the high degree of
purification of the two synthesized triphosphate nucleotides
As expected,1H NMR measurements of both GTPcNH2
and GTPcF in aqueous solution at 20C, pH 7.4 showed
no difference to the natural substrate GTP [19] as the
guanine base is not affected by the modifications and as
the c-phosphate amide hydrogens are in fast exchange with
the solvent In Fig 2 31P NMR spectra are shown for
GTPcNH2 and GTPcF, and their respective metal ion
complexes with Mg2+
For GTPcNH2 the appearance of three discrete
reson-ance lines with similar intensity confirms the uniformity and
the conformational identity of the substrate The observed
mean half width of, e.g 4.7 Hz for the c-resonance line is
typical for a molecule of 523 Da mass at 20C in aqueous
solution The resonance lines could be assigned by their J-coupling constants and by comparison to unmodified GTP While the chemical shift of the a-phosphate group changed only little upfield compared to GTP, the b-phosphate was shifted upfield by about)1.5 p.p.m and the terminal c-phosphate shifted by almost 5 p.p.m down-field by the replacement of the hydroxy OH–with an amide
NH2 Complexation of GTPcNH2 with Mg2+led to an additional downfield shift of all phosphate groups, with the b-phosphate changing most This observation was similar
to the change in GTP when coordinated with magnesium, but the absolute shift change was almost 1 p.p.m smaller (from 1.26 p.p.m to 2.21 p.p.m.) than in the natural substrate The2JPP-coupling constants of GTPcNH2 and GTPcNH2Mg2+ analogues showed smaller alterations when compared to GTP In both cases the b-phosphate groups appeared as triplets as the coupling constants between
Pa–Pband Pb–Pcwere almost identical, while coordination with magnesium again decreased the coupling constants
In GTPcF four phosphorus lines appeared as the coupling between the natural spin ½ nuclei 31P and 19F led to a splitting of the terminal phosphate resonance This direct coupling constant1JPcFwas about 936 Hz and hardly changed upon magnesium coordination (934 Hz), indica-ting a strong interaction between the two nuclei Chemical shift changes of GTPcF compared to GTP were much more distinct than for GTPcNH2 All three phosphate groups shifted upfield; in the case of the c-phosphate the shift was )12.6 p.p.m By contrast, coordination to magnesium caused only slight chemical shift changes, of which the
Fig 2. 31P NMR spectra of G TPcNH 2 (A) and GTPcF (B) (top) and their respective magnesium ion complexes (bottom) Spectra were recorded at pH 7.4 and 20 C in aqueous solution.
Trang 5largest was)0.5 p.p.m for the c-phosphate This might be
an effect of the low magnesium binding affinity, as will be
discussed later A similar observation was made for the19F
NMR resonance line at position 0.91 p.p.m which changed
only to 0.97 p.p.m upon magnesium saturation Finally,
the J-coupling values between the three phosphates again
tended to be very insensitive to modifications, and fell by
around 25% on complexation with magnesium All
chem-ical shift data and J-coupling constants reported are
summarized in Tables 1 and 2
Nucleotide stability
We next tested the stability of the GTPcNH2 nucleotide
derivative In 0.1Mtriethanolamine/HCl buffer at pH 7.6
the spontaneous hydrolysis of GTPcNH2 at room
tem-perature was less than 1% in five days In contrast, at
pH 4.5 in 0.1Mpotassium phosphate buffer the nucleotide
was hydrolysed to GDP with a half time of about 48 h
Titration of GTPcNH2with HCl/NaOH monitored by31P
NMR spectroscopy showed no variation of the chemical
shifts of the three-fold negatively charged phosphate groups
from pH 3 to pH 11 At pH 2.8 the intrinsic hydrolysis
increased (so called acidic hydrolysis) and GTPcNH23–was
rapidly transformed to GDP3–+ H2PO4 + NH4+by two
water molecules The intermediate compound
phosphor-acid-amidate H2PO3NH2 was not observed by NMR
As a control, we titrated H2PO3NH2 in the range from
pH 11 to pH 1.8 The 31P chemical shifts for the three
different protonation states were found to H2PO3NH2
at )6.90 p.p.m., [HPO3NH2]– at )2.65 p.p.m., and
[PO3NH2]2–at +7.97 p.p.m The pKavalues between these
three states were determined to pK(0/1–)¼ 3.02 ± 0.05 and
pK(1–/2–)¼ 8.46 ± 0.02 using a least square fit to 15
individual measured chemical shift values (data not shown)
Since the resonance lines for the a-and b-phosphate groups of
GDP at pH 2.8 were located at)10.73 and )10.20 p.p.m.,
respectively, a possible signal overlap between GTPcNH2,
GDP, HPO3NH2and H3PO4 (Pi) could be excluded We
therefore assume that at low pH (pH < 3) GTPcNH2 is
first transformed to ammonia and GTP, the latter being
subsequently hydrolysed to GDP and Pi
Magnesium binding and magnesium exchange rates
To analyse the metal ion binding properties of GTPcNH2
we first performed a magnesium titration series and a
temperature series by NMR spectroscopy Complete
line-shape analysis simulations of the complex formation of
GTPcNH2 with Mg2+were performed on the entire 31P
NMR spectra (a-, b-and c-phosphorus nuclei) and showed
a reasonably good agreement for all three resonance lines
This is demonstrated in Fig 3 where the part of the NMR
spectra and simulations that show the b-phosphate is
displayed The b-resonance line underwent the biggest
resonance shift and was therefore most sensitive to changes
in the exchange rate, as the titration with magnesium from
null to complete saturation indicates (Fig 3)
Next, we determined the binding energy of GTPcNH2to
magnesium by a complete lineshape analysis of a series of
NMR spectra We adjusted the saturation of GTPcNH2
with Mg2+to 45% and varied the temperature from 5C to
65C in 13 steps of 5 Five representative31P NMR spectra
of the b-resonance line and the corresponding simulations are shown (Fig 4) The fitted exchange rates in aqueous solutions ranged from 900 to 9000 Hz with relative margins from ± 22% at 5C to ± 8% at 30 C As the plot against reciprocal temperature shows, the simulated exchange rates k nicely fit to the Arrhenius equation
k¼ k0exp(–DH/RT) with R the gas constant and T the absolute temperature (Fig 5) Based on these values the activation energy DH for the GTPcNH2Mg2+ complex formation was determined to be 7.8 ± 0.15 kcalÆmol)1 This result is similar to the activation energy for magnesium binding of the natural substrate ATP which has been determined to be 8.1 kcalÆmol)1[20]
Association of magnesium ions with different nucleotides
In biological systems it is the complex between the nucleotide and the magnesium ion which is bound to an ATP or GTP binding enzyme rather than the nucleotide only Therefore, ITC was employed to quantify the interaction between the nucleotides and the magnesium ion (Fig 6 and Table 3) As expected all nucleotides bound one magnesium ion as indicated by the stoichiometry factor
N¼ 1 (Table 3) Basically, for all complex formation reactions an unfavorable enthalpy change was observed, which was counteracted by a TDS value two to three times
as large In comparison to GTP the affinity for the magnesium ion was lower for GTPcNH2 and GTPcF For GTPcS the association constant was only two-fold smaller whereas for GTPcNH2and GTPcF this constant was significantly smaller, namely 34-fold and 140-fold, respectively Most probably this is due to the decreased negative charge at the c-position in GTPcNH and GTPcF
Fig 3.31P NMR spectra of a MgCl 2 titration series added in increasing amount to GTPcNH 2 The resonance line of the b-phosphate in the experimental measurements (left) and its corresponding simulation (right) are shown Note the shift and the intermediate broadening of the resonance line The amount of GTPcNH 2 Mg 2+ complexes relative
to free GTPcNH 2 nucleotide is indicated on the left The determined exchange rates based on the exchanging spin system simulation are shown right The spectra were measured at 20 C and pH 7.4 in aqueous solution.
Trang 6where the protic hydroxy group is replaced by the amino
and fluoride groups, respectively In contrast, the sulfur in
GTPcS may take on the role of the oxo-group It should be
noted that the smaller affinities of GTPcNH and GTPcF
are predominantly due to lower DS values, possibly reflecting the release of less water into bulk upon complex formation
Application of the nucleotides to the GTPase Ras The suitability of the two nucleotide analogues for biolo-gical macromolecules was finally tested using the small GTP-binding protein Ras (reviewed in [21,22]) We success-fully loaded the nucleotide analogues onto the 21 kDa GTPase Ras using the alkaline phosphatase method, which yielded a tightly bound protein-nucleotide complexes, as found for RasÆGTP [23] First the intrinsic GTPase rate of wild-type H-Ras (1–167) complexed with magnesium ions and various guanosine triphosphate nucleotide analogues was determined by HPLC measurement (Table 4) At 37C the intrinsic hydrolysis rate of Ras-bound fluorotriphos-phate GTPcF was only twofold lower than for the natural
Fig 5 Arrhenius plot of the simulated magnesium ion exchange rate
constants (k) vs the reciprocal absolute temperature (1/T ) for
GTPcNH 2 The activation energy DHfor GTPcNH 2 ÆMg 2+ complex
formation is determined to 7.8 ± 0.15 kcalÆmol)1.
Fig 6 Isothermal titration calorimetry of GTPcNH 2 with MgCl 2 To a solution of 5.0 m M GTPcNH 2 placed in the cell of the calorimeter a solution of 50 m M MgCl 2 was injected in steps of 6 lL each (the first step was 2 lL only) The increase in heating power was detected (upper panel) The power pulses were integrated and plotted vs the molar ratio of injected MgCl 2 and nucleotide (lower panel) A fit to the experimental data yields the stoichiometry factor N ¼ 0.96, the association constant K a ¼ 0.82 m M )1 and the enthalpy of association DH ¼ 3.9 kcalÆmol)1.
Table 3 Thermodynamic parameters for the association of magnesium ions with different nucleotides obtained by isothermal titration calori-metry DS is calculated according to the Gibbs-Helmholtz equation.
Nucleotide
N (Nucl./Mg)
(mol/mol)
K a
(m M )1 )
DH
(kcalÆmol)1)
DS
(calÆmol)1ÆK)1)
Fig 4.31P NMR spectra of a temperature series of GTPcNH 2 Mg2+.
The b-phosphate resonance line at )22.19 p.p.m is shown in an
intermediate exchange state at 45% Mg2+saturation Experimental
measurements (left) and simulated spectra (right) are displayed
showing temperature values and the simulated exchange rates,
respectively Lyophilized GTPcNH 2 was dissolved to 2.1 m M
con-centration in aqueous solution and adjusted to pH 7.4 with HCl/
NaOH MgCl 2 was added to 1 m M concentration The precise
saturation was determined from the chemical shift position at 20 C
(see Fig 3 and Table 1).
Trang 7substrate GTP while the rate for the thiotriphosphate
GTPcS was about 11-fold lower Most stable with up to
190-fold lower hydrolysis rates were the two triphosphate
analogues with b,c-substitutions GppCH2p and GppNHp
The Ras-catalysed hydrolysis rate of GTPcNH2finally lay
midway between the rates for GTPcS and GppNHp, with a
3-fold difference to both
The more stable RasÆGTPcNH2ÆMg2+ complex was
subsequently studied by31P NMR spectroscopy A
partic-ular feature of the Ras protein is the flexibility of the effector
loop which can be detected in the triphosphate bound form
by a line splitting of the phosphorus resonances [24] The
exchange is due to at least two distinct conformations which
can be observed also by heteronuclear NMR [25,26] or in different crystal forms of Ras protein [27,28] Flexibility in the active center of G-proteins has been also observed for RanGTP [29] and in different conformations of the switch regions in the crystal structures of Rap2A complexed with GTP, GDP and GTPcS [30] As shown in Fig 7 (bottom spectrum) this feature was preserved for Ras bound to GTPcNH2 At low temperature (5C) the b-phosphate resonance was split into a less populated high field shifted peak (b1, 27%) and a highly populated down field shifted peak (b2, 73%) A temperature series from 2 C to 30 C revealed the coalescence of both lines at approximately
15C which is typical for a two-site exchange with a transition from slow to fast exchange (data not shown) As described for the intrinsic hydrolysis of GTPcNH2, the Ras-catalysed hydrolysis of bound GTPcNH2did not lead to the observable formation of the compound H2PO3NH2in the NMR spectra (which is expected at)2.7 p.p.m.) Instead, the resonance signals for Piand Ras-bound GDP increased during the time course of the experiment (Fig 7, compare bottom and top spectra) suggesting the formation of ammonia and Ras-bound GTP before hydrolysis
A concentration series with the effector protein Raf-RBD
at 5C added in increasing amount from 0.2 to 1 molar ratio showed the progressive stabilization of one particular conformation due to its high affinity for triphosphate bound Ras (Fig 7) Most remarkably, also the c-phosphate group
is perturbed by this interaction and shifted about )0.8 p.p.m upfield (Table 5) These data indicate the ability
of GTPcNH2 to function as a triphosphate nucleotide analogue with characteristic properties
D I S C U S S I O N The data reported here demonstrate the synthesis of the two guanosine triphosphate nucleotide analogues GTPcNH2 and GTPcF, their biochemical characterization and appli-cation to the GTP-binding protein Ras GTPcNH2 was prepared by the method described by Knorre et al [31] for ATP derivatives c-Amide derivatives of GTP were des-cribed by Babkina et al who used, e.g the c-(4-azido) anilide of GTP to substitute efficiently for GTP as a photoaffinity label in the elongation factor protein EF-Tu [32] The GTPcF substrate analogue was first prepared by Eckstein et al [9], by the method of Haley & Yount [33], and used to study its interaction with the GTP-binding site
of adenylyl cyclase [34] We synthesized this substance by
Table 4 Intrinsic GTPase rate of wild-type H-Ras(1)167)Mg 2+ protein
at 37 °C bound to various guanosine triphosphate nucleotide analogues.
Hydrolysis rates were determined with HPLC by measuring the
con-centration of protein-bound tri- and diphosphate nucleotides Buffer
conditions: 25 m M Tris/HCl at pH 7.4, 2.5 m M MgCl 2 and 1 m M
DTE.
Nucleotide GTPase rate (10)5min)1)
Fig 7.31P NMR spectra of protein bound RasÆGTPcNH 2ÆMg2+and
concentration series with Raf-RBD The ratio of Raf-RBD to Ras
varies from 0 (bottom spectrum) to 1 (top spectrum) as indicated on
the right Spectra were recorded at pH 7.4 and 5 C in 25 m M Tris/
HCl buffer, 2.5 m M MgCl 2 and 1 m M dithioerythritol Note the
splitting of the b-phosphate resonance into two states for
RasÆGTPcNH 2 ÆMg2+(bottom spectrum) and the stabilization of one
conformation upon complexation with Raf Excess of free and bound
phosphate groups are labelled.
Table 5 31 P chemical shifts of Ras(1)167)ÆGTPcNH 2ÆMg 2+ at pH 7.4,
5 °C Spectra were recorded in 25 m M Tris/HCl buffer, 2.5 m M Mg 2+
and 1 m M DTE The splitting of the a-and b-phosphate resonance lines in protein bound triphosphate-nucleotides is a specific feature of the Ras protein, indicating different conformations of the active center [24].
31
P chemical shift (p.p.m.) Proteinnucleotide a (1) a (2) b (1) b (2) c RasGTPcNH 2 Mg2+ – )11.80 )16.15 )16.85 1.90 RafRasGTPcNH 2 Mg 2+ – )11.76 – )16.81 1.07 RasGppNHpMg2+ )11.15 )11.85 )2.69 )3.41 )0.32
Trang 8the simple method of Wittmann [10], which works very well
with adenine nucleotides The presence of a guanine base
gives rise to the formation of yellow by-products, probably
due to the reaction of 2,4-dinitrofluorobenzene with the
amino group of GTP Thus in this case the simplicity of the
method is at the expense of yield
The kinetic parameters determined reveal the
biochemi-cal properties of the two nucleotide analogues While the
activation energy of magnesium binding for GTPcNH2is
similar to that of the natural substrate [20,35], the
association constant Ka for magnesium is significantly
smaller for both nucleotides analysed Therefore, the more
stable c-amido triphosphate analogue may be particularly
useful for the study of the role of divalent cation binding,
e.g to analyse a proposed reaction mechanism For
example, the influence of the magnesium binding affinity
on the kinetics of the Ras guanine nucleotide exchange
factor Sos [36] can be studied with the nucleoside
diphosphate GDPcNH2 derivative loaded onto Ras
Additionally, an intermediate magnesium-free state may
be stabilized more easily with GTPcNH2 or ATPcNH2
analogues The analogue ATPcNH2 may provide new
insights into the equilibrium between different
conforma-tions of myosin [37] Finally, specific labeling of the amide
group with 15N isotopes may be useful for nitrogen
selective heteronuclear NOE experiments for the structural
analysis of the active center in solution In combination
with specific labeling of single residues in the protein [38]
this may yield detailed insights into the dynamics of the
nucleotide binding site
The GTPcF analogue may be particularly useful
because of the high sensitivity of19F NMR spectroscopy
In a previous report the GTP binding site of the 110 kDa
protein tubulin was studied using the fluorotriphosphate
[39] Here, fluorine relaxation rates were determined to
analyse the location of the divalent cation site relative to
the exchangable nucleotide We are analysing the
possi-bility of two distinct nucleotide binding sites in the human
guanylate-binding protein 1 (hGBP1) [40] by titration of
GTPcF or GDPbF to the noncomplexed protein,
assu-ming different chemical environments for each putative
nucleotide binding site (data not shown) Finally, the
suitability of both nucleotide derivatives for the use in
solid state NMR spectroscopy should be tested in future
experiments
For the slowly hydrolysing Ras protein the two
nucleotides described here close the 10-fold gap in intrinsic
hydrolysis rates between bound GTP and GTPcS, and
GTPcS and GppNHp (Table 4) This broad variety allows
an almost individual selection of hydrolysis stability from
protein bound GTP to protein bound GppCH2p for all
different purposes The application onto the GTP-binding
protein Ras confirmed that the flexibility of the effector
loop of Ras is preserved (Fig 7) For nucleotide binding
proteins the flexibility of the nucleotide binding site in its
active center is a vital prerequisite for the dynamic
function of the protein This is particularly important for
mechanistic studies and the analysis of binding
intermedi-ates, as shown for the complex formation of Ras with the
effector protein Raf-RBD [24,41] With these two
deriv-atives characterized we extend the variety of nucleotide
analogues available for kinetic, structural, and cellular
studies
A C K N O W L E D G E M E N T S
We thank John Wray and Roger S Goody for discussions and Ulrich Haeberlen, Alfred Wittinghofer and Kenneth C Holmes for continu-ous support M.G acknowledges support by the Peter und Traudl Engelhorn Stiftung (Penzberg, Germany).
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