10308
Biochemistry 1996, 35, 10308-10320
Conformational Transitions in p21ras and in Its Complexes with the Effector Protein Raf-RBD and the GTPase Activating Protein GAP† Matthias Geyer,‡ Thomas Schweins,§ Christian Herrmann,§ Thomas Prisner,| Alfred Wittinghofer,§ and Hans Robert Kalbitzer*,‡ Max-Planck-Institut fu¨ r medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Max-Planck-Institut fu¨ r molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, and Freie UniVersita¨ t Berlin, Fachbereich Physik, Arnimallee 14, 14095 Berlin, Germany ReceiVed December 4, 1995; ReVised Manuscript ReceiVed April 12, 1996X
ABSTRACT: 31P
NMR revealed that the complex of p21ras with the GTP analog GppNHp‚Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy ∆Hq and activation entropy ∆Sq for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wildtype protein one obtains ∆Hq ) 89 ( 2 kJ mol-1 and ∆Sq ) 102 ( 20 J mol-1 K-1 and for the mutant protein ∆Hq ) 93 ( 7 kJ mol-1 and ∆Sq ) 138 ( 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggests that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (95% as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The tightly bound nucleotide GDP was exchanged for the slowly hydrolyzing GTP analog GppNHp according to John et al. (1990). Excess nucleotide and phosphate ions were removed by gel filtration. The protein concentration was determined with the Bradford assay using bovine serum albumin as standard (Bradford, 1976). The amount of protein-bound nucleotide was analyzed with C18 reverse-phase HPLC and quantitated with a calibrated detector (Beckman) and integrator (Shimazu), respectively. GAP-334 (Gapette) was expressed in E. coli and purified as described before (Gideon et al., 1992). The Ras-binding domain of human c-Raf-1 (Raf-RBD, 81 residues) was expressed in E. coli and purified as described recently (Herrmann et al., 1995). Preparation of the NMR Samples. 31P NMR spectra of p21ras complexed with G nucleotides were recorded in 40 mM Hepes, 10 mM MgCl2, and 0.1 mM DTE. Usually, 2.5 mL of the sample was placed in 10 mm NMR tubes (Wilmad) with a protein concentration of 0.4-1.2 mM. To provide a lock signal, the samples contained between 10% and 99% D2O. For some experiments the sample had to be freeze-dried and redissolved in D2O. Here, care was taken that the sample was kept permanently at low temperature (liquid nitrogen) and that the freeze-drying process was as short as possible. The protein-free nucleotide samples contained approximately 10 mM nucleotide (GDP, GTP, or GppNHp) in the above buffer but with 30 mM MgCl2 in solution. For experiments on p21ras complexes with GAP or Raf-RBD, usually 1 mM p21ras and highly concentrated GAP (0.7 mM) or Raf-RBD (3.4 mM) solutions were added. NMR Spectroscopy. 31P and 1H NMR experiments were performed on a Bruker AMX-500 NMR spectrometer working at a proton resonance frequency of 500 MHz and a phosphorus resonance frequency of 202 MHz. Additional phosphorus spectra were recorded on a Bruker AMX-600 and a Bruker HX-360 spectrometer at 243 and 146 MHz, respectively. The 31P spectra were referenced to 85% phosphoric acid contained in a glass sphere which was immersed in the sample and calibrated for various temperatures. Unless noted otherwise, phosphorus spectra were collected at 5 °C with a total spectral width of 60 ppm (100 ppm for samples containing GTPγS). For one-dimensional 31P NMR spectra, 512-8192 free induction decays were summed after excitation with a 65° pulse using a repetition time of 3-5 s. Most experiments were run in D2O solution in order to suppress undesired proton phosphorus couplings. A total of 32 K time domain data points were recorded and transformed to 16K real data points corresponding to a digital resolution of 0.74 Hz/point. 1H NMR spectra were recorded with selective presaturation of the water resonance for 1.5 s. All spectra were processed on a Bruker X32/3 workstation with the UXNMR standard software. Spectra used for the complete line-shape analysis were filtered by an exponential window function causing no significant line broadening. The spin-spin coupling constants of the nucleotide-Mg2+ complexes were determined from a line fit (see below) of a nonfiltered 1D spectrum with a digital resolution of 0.25 Hz/point after Fourier transformation.
10310 Biochemistry, Vol. 35, No. 32, 1996
Geyer et al.
Preparation of the EPR Samples and EPR Spectroscopy. The p21ras‚GppNHp‚Mn2+ complex was generated by incubation of p21‚GppNHp‚Mg2+ with 40 mM ammonium sulfate and 100 mM MnCl2 for about 60 min. Excess metal ions were removed by gel filtration with PD-10 columns (Pharmacia). All buffers used were equilibrated with a chelating resin (Chelex 100, Bio-Rad) to remove traces of bivalent metal ions. DTE (10 mM) was added to the buffer to prevent oxidation of the protein and of Mn2+. To ensure that the Mg2+ ion is replaced quantitatively by Mn2+, the exchange process was repeated twice. EPR spectra were recorded with a home-built high-field spectrometer operating at 95 GHz and 3.4 T, respectively (Prisner et al., 1994). The spectra were measured in the cw mode with a microwave power of 1 µW and a modulation amplitude of 0.1 mT. Protein concentration was in the range 1.0-1.5 mM in 50 mM Tris-HCl buffer of pH 7.4. Determination of Exchange Rates. The NMR spectra were evaluated with a simulation software written in our laboratory. The software package is written in C and runs on UNIX workstations. The simulated spectra were displayed and plotted with the program UXNMR from Bruker, Karlsruhe. The two-site exchange was simulated on the basis of the full-density matrix formalism as described by Kaplan & Fraenkel (1980) and Nageswara Rao (1989). For triphosphate nucleotides the system can be described as k1
} A′B′C′ ABC {\ 1 k-1 2
(1)
1 T2x
with ABC the R-, β-, and γ-phosphate in state 1 and A′B′C′ the phosphate groups in state 2 and k1 and k-1 the rate constants for this process. For a description of this process the populations p1 and p2 of states 1 and 2 must be known. The equilibrium constant K is given by
K ) k1/k-1 ) p2/p1
much with the temperature and can be considered as constant for the following line-shape analysis. The most difficult parameters to obtain are the transverse relaxation times T2 since they are temperature dependent and since it is difficult to determine them in the presence of exchange broadening. In principle, they can be obtained for extreme temperatures with |2π ∆ν| . 1/τe or |2π ∆ν| , 1/τe directly from the line widths. This method, which is commonly applied in physical chemistry, fails in our case since the extreme temperatures necessary cannot be obtained without denaturation of the protein or freezing of the sample. However, a lower limit on the T2’s can be obtained with the assumption that at the lowest temperature measured exchange broadening is negligible. Since the transverse relaxation time should increase with temperature (assuming relaxation via dipolar interaction and chemical shift anisotropy in a rigid body model), the lower limit obtained holds for the whole temperature range. The principal functional form of the temperature dependence of the relaxation rates depends on the relaxation mechanisms involved. Since this temperature dependence represents only a small correction term in the calculation of the exchange rates, it is sufficient to assume only relaxation via dipolar interaction and chemical shift anisotropy and isotropic rotational reorientation with the correlation time τr. The transverse relaxation rates for the phosphorus nucleus x (x ) A, B, C, A′, B′, C′) can be written as
(2)
NMR parameters entering the calculation are the spin I ) 1/ of 31P, the resonance frequences ν , ν , ν , ν , ν , and 2 A B C A′ B′ νC′, the coupling constants JAB, JBC, JAC, JA′B′, JB′C′, and JA′C′, and the transverse relaxation times T2,A, T2,B, T2,C, T2,A′, T2,B′, T2,C′. The J-coupling constants in the protein-bound state cannot be obtained directly, since they are not resolved in the spectra. They were approximated by the J-couplings in the corresponding free nucleotide-Mg2+ complexes which can be obtained experimentally. The small changes in these constants that are possible in the bound state can only marginally influence the observed results. The 31P resonance frequencies were obtained by fitting the corresponding p21nucleotide spectrum recorded at the lowest temperature where slow exchange conditions prevail. Here, a gradient fit procedure, using the Marquardt method [see, e.g., Press et al. (1986)], was performed to determine the mean half-width, the signal amplitude, and the line position of the overlapping peaks by assumption of the J-coupling constants and Lorentzian lineshapes. From these parameters the populations p1 and p2 in the different states can be derived. As is usual for the simulation of temperature dependence of exchange spectra, it was assumed that the J-couplings, the equilibrium constant K, and the resonance frequencies νj do not vary
)
µ0 p2γx2 4π2 20
∑
γj2
j*x r 6 xj
fDD(τr,ωx,ωj) + 2
γxB02∆σ2fCSA(τr,ωx) (3)
15
with µ0 the vacuum permeability, γx the phosphorus magnetogyric ratio, γj the proton or phosphorus magnetogyric ratio, and rxj the distance between spins x and j. For proteins of the size of p21ras, functions fDD and fCSA can be assumed to be approximately proportional to τr. As long as the environment of the spin x does not change with the temperature, eq 3 can be simplified to
1 ) cxτr T2x
(4)
with cx a constant characterizing the environment of spin x. For isotropic rotation τr is given by the Stokes-Einstein relation
η τr ) V′ kT
(5)
with η the viscosity of the solution and V′ the effective volume of the molecule. The temperature dependence of the viscosity of aqueous solution can be described by
η ) Ae-b/T
(6)
with A and b empirical constants. These constants were determined from viscosity data of H2O and D2O tabulated, e.g., in Landolt-Bo¨rnstein (1969). A fit to the data in the range from 273 K (e.g., 277 K) to 313 K results in A ) 1.737 g m-1 s-1, b ) 0.0262 K for H2O and A ) 2.279 g m-1 s-1, b ) 0.0285 K for D2O. From eqs 4-6 it follows
Conformational Transitions in p21ras
Biochemistry, Vol. 35, No. 32, 1996 10311
that
1 V′A ) cx e-b/T kT T2x
(7)
The simulation of the spectra and the calculation of the parameters were performed iteratively. First, for the lowest temperature spectrum the upper limit of the exchange rate kmax was determined for slow exchange. For a grid of rate constants in the range between 0 and kmax the transverse relaxation times for the different resonances were calculated. Starting with these values the complete temperature series was fitted assuming the temperature dependence given by eq 7. By visual inspection of the measured and simulated spectra the range of possible exchange rates at the lowest temperature could be narrowed down considerably. Within this range the simulation parameters were refined iteratively until the optimal solution had been found. The temperature dependence of the rate constants was fitted with the program Grafit (Erithacus Software) on the basis of transition state theory (Eyring, 1935) to the equation q q kT k1 ) κ e-(∆H -T∆S )/RT h
(8)
where κ is the transmission coefficient, k the Boltzmann constant, h the Planck constant, R the gas constant, T the absolute temperature, ∆Hq the activation enthalpy, and ∆Sq the activation entropy. RESULTS 31P NMR Spectra of the p21‚GppNHp‚Mg2+ Complex. The phosphorus NMR spectra of triphosphates bound to proteins with a single nucleotide binding site usually show only three resonances, corresponding to the R-, β-, and γ-groups of the complexed nucleotide. Published 31P NMR spectra of p21-nucleoside triphosphate complexes (Klaus et al., 1986; Ro¨sch et al., 1986; John et al., 1989; CampbellBurk, 1989; Grand et al., 1989; Franken et al., 1993) as well as the spectra we recorded at room temperature correspond to this scheme. In contrast, additional phosphorus resonance lines become visible in the spectra of the p21‚GppNHp‚Mg2+ complex at lower temperatures (Figure 1). The line widths and line positions exclude the possibility that these lines arise
FIGURE 1: 31P NMR spectra of p21ras‚GppNHp‚Mg2+ at different temperatures: 0.7 mM wild-type p21ras complexed with GppNHp and 2.0 mM MgCl2 in D2O, pH 7.4. (Bottom) Spectrum at 0 °C, recorded before the high-temperature spectrum (Middle) of 30 °C. (Top) Temperature 0 °C, recorded after the high-temperature spectrum.
from free nucleotide. Therefore, they must result from different states, states 1 and 2, of the nucleotide bound to the protein. A closer inspection suggests that the resonance lines of the different phosphate groups split in two lines. This is most obvious for the resonance of the β-phosphate but is also visible as inhomogeneity of the R-phosphate line. Only the γ-phosphate line appears to be unperturbed. A careful integration of fully relaxed spectra shows that the integral of the γ-phosphate line equals that of the sum of the two β-phosphate lines or the R-phosphate lines. The data are consistent with the assumption of two states of the nucleotide-protein complex with R-resonances at slightly different positions, β-lines at clearly different positions, and γ-lines at the same positions. An increase in temperature leads to a coalescence of lines as is typical for a two-site exchange with a transition from slow exchange to fast exchange. This process is completely reversible, the initial spectrum essentially returning when the temperature was lowered (Figure 1). This behavior also excludes the possibility that one of the two states corresponds to nucleotide binding to irreversibly denatured protein. A possible explanation for the observation of the two states could be that, under our experimental conditions, the nucleotide is complexed with magnesium in one of the states and not in the other. Although this is very unlikely from the known binding constants for divalent ions to p21, it was checked experimentally. In the range of 0.1-100 mM free Mg2+ in solution no spectral changes were observed. This excludes the possibility that the partial occupancy of the highaffinity site of p21 or a possible low-affinity binding site for Mg2+ is responsible for the observed line splitting. The line splitting and the relative intensities (populations) of the lines are not dependent on pH in the range from 6.5 to 10.4. 31P NMR Spectra of the p21‚GppNHp‚Mg2+ Complex in Mutant Proteins. To check whether the observed conformational equilibrium is a special property of wild-type
FIGURE 2: 31P NMR spectra of wild-type and mutant p21ras complexes with GppNHp‚Mg2+. Spectra were recorded at 5 °C and pH 7.4. Protein concentrations were wild type, 0.7 mM; S65P, 0.8 mM; G12D, 0.7 mM; Y32R, 0.4 mM; T35A, 0.4 mM; all with 10 mM free Mg2+. Note that the number of scans for each spectrum varied.
10312 Biochemistry, Vol. 35, No. 32, 1996 Table 1:
31P
Geyer et al.
NMR Chemical Shifts of Different p21ras-Nucleoside Triphosphate Complexesa chemical shifts δ (ppm)b
complex
R(1)
R(2)
β(1)
p21(wt)‚GppNHp‚Mg2+ p21(G12D)‚GppNHp‚Mg2+ p21(Y32R)‚GppNHp‚Mg2+ p21(T35A)‚GppNHp‚Mg2+ p21(E62H)‚GppNHp‚Mg2+ p21(S65P)‚GppNHp‚Mg2+
-11.15 -11.49 -10.98 -11.09 -11.21 -11.28
-11.85
-2.69 -2.47 -2.53 -2.51 -2.60 -2.58
-10.85
p21(S65P)‚GppNHp p21(wt)‚GTP‚Mg2+ p21(G12V)‚GTP‚Mg2+ p21(Q61H)‚GTP‚Mg2+ p21(Q61L)‚GTP‚Mg2+ p21(Q61P)‚GTP‚Mg2+ p21(S65P)‚GTP‚Mg2+
-11.82 -11.78
-11.77 -11.76 -11.84 -11.67 -11.71 -11.74
β(2) -3.41 -3.39 -3.42 -3.59
γ(1)
γ(2)
-0.41 -0.36 -0.23 -0.25 -0.38 -0.37
-0.23
-3.95 -14.97 -14.94 -14.93 -14.70 -15.10 -14.96
-1.45
∼-15.7 -15.73 -15.81
p21(Q61H)‚GTP
-11.27
-16.53
p21(wt)‚GTPγS‚Mg2+
-11.30
-16.62
-0.24 -0.23
-7.99 -7.72 -7.81 -7.73 -7.75 -8.31
equilibrium c constant K(12) 1 1.09 0.52
