Biochemistry 1981, 20, 6265-6272
6265
Spectroscopic Studies of the Nucleotide Binding Site of Elongation Factor Tu from Escherichia coli. An Approach to Characterizing the Elementary Steps of the Elongation Cycle of Protein Biosynthesist John F. Eccleston
9-~-Ribofuranosyl-2-amino-6-mercaptopurine of thioGDP and thioGTP to EF-Tu to be determined by stopped-flow spectrophotometry in the presence of 5 mM 5’-diphosphate (thioGDP), thioGTP, and 2-azidoinosine 5’MgC12, at 20 OC and pH 7.6. Values of 1.1 X los and 1.6 diphosphate have been shown to bind to elongation factor Tu X IO4 M-’ s-l, respectively, were obtained. Similarly, values (EF-Tu) from Escherichia coli. The binding of thioGDP of the dissociation rate constants were 0.01 s-l for thioGDP results in a 4-nm red shift of the absorption band of thioGDP, and 0.21 s-l for thioGTP. The dissociation rate constant of an increase of approximately 0.7 pH unit of the pK of the thiol Mg from EF-Tu-MgthioGDP was found to be 1.4 s-’, showing group, and the formation of a circular dichroism spectrum of that Mg can dissociate independently of the nucleotide. The the nucleotide distinctly different from that of the free nuspectroscopic and kinetic data show that the nucleotide binding cleotide. These results are consistent with the purine moiety site of EF-Tu has some properties similar to that of myosin being bound in a highly hydrophobic site of EF-Tu with but also some marked differences. ThioGTP substitutes for specific interactions of the thione group with the protein. GTP in the EF-Tu-dependent binding of aminoacyl-tRNA to When thioGTP binds to EF-Tu, the red shift of the spectrum ribosomes, and the dissociation of thioGDP from EF-Tu is is only 2 nm, the pK of the thiol group is increased by 0.1 pH catalyzed by elongation factor Ts. The potential of thioGTP unit, and the CD spectrum is less intense. This indicates that the nucleotide binding site is less hydrophobic in EF-Tw for investigating the kinetic steps of the elongation cycle of thioGTP as compared to EF-TwthioGDP. The spectral protein biosynthesis by stopped-flow spectrophotometry is perturbations have allowed the rate constants for the binding discussed. ABSTRACT:
%e prokaryotic elongation factor Tu (EF-Tu)’ is involved in the elongation cycle of protein biosynthesis by forming a ternary complex with GTP and aminoacyl-tRNA which then binds to the ribosome containing mRNA. GTP is hydrolyzed, the aminoacyl-tRNA is positioned at the A site prior to subsequent peptide bond formation, and a binary complex of EF-TwGDP is released [reviewed by Miller & Weissbach (1977)l. In order to understand the role of EF-Tu and GTP in the fast and accurate incorporation of amino acids into the growing peptide chain, we are investigating the elementary steps of the elongation cycle by using rapid reaction kinetic techniques combined with single turnover conditions which have given important information about the mechanism of other energy-transducing nucleoside 5’-triphosphatases (Trentham et al., 1976). Steps involving covalent bond breaking or formation can be followed by chemical quenching, and the rate of GTP hydrolysis and peptide bond formation has been measured when Phe-tRNA-EF-TwGTP binds to poly(U)-programmed ribosomes containing acetyl-Phe-tRNA in the P site (Thompson et al., 1980). Steps involving binding, rearrangements, or product release are more difficult to study but can be followed by intrinsic or extrinsic spectral probes. For this reason, I have investigated the binding of potential chromophoric and fluorescent analogues of guanosine nucleotides to EF-Tu. EF-Tu is a globular protein with a molecular weight of 43 225. Its primary sequence has been determined (Arai et al., 1980), and a high-resolution (2.6-A) X-ray diffraction analysis has been made on a modified form of the EF-TwGDP complex (Morikawa et al., 1978). EF-Tu binds GDP with a Kd of 3 X lo-’ M and GTP with a Kd of 3 X lo-’ M, both From the Department of Biochemistry and Biophysics, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19 104. Received March 18, 1981. The work was supported by grants from the American Cancer Society (NP 312) and the Whitehall Foundation.
measurements being at 20 OC and pH 7.4 in the presence of 5 mM MgC12 (Miller & Weissbach, 1977). The major functional difference between EF-TwGTP and EF-TwGDP is that the former complex binds aminoacyl-tRNA (Kd 10-8 M) whereas EF-TwGDP does not bind aminoacyl-tRNA [or M (Shulman et al., if it does, with a Kd of greater than 1974)], Conformational differences between EF-TwGTP and EF-TwGDP have been studied by a variety of techniques [reviewed by Kaziro (1978)l. Thioguanosine and its corresponding nucleotides (Figure 1) have an absorption maximum at 342 nm enabling accurate spectral measurements to be made in the presence of protein. A weak negative CD peak is associated with this absorption band. The 6-mercapto group ionizes with a pK in the region of 8.2-8.6, the resulting anionic form having an absorption maximum at 320 nm. The absorption spectrum, pK, and CD spectrum of thioGTP and thioGDP are perturbed when these nucleotides bind to myosin, and the absorption changes have been used to investigate the nucleotide binding site of myosin and determine the kinetic constants of the elementary steps of the myosin ATPase mechanism (Eccleston & Trentham, 1977, 1979; Eccleston & Bayley, 1980). Potential fluorescent analogues of guanosine nucleotides are derived from 2-azidoinosine and 9-P-ribofuranosyl-2-aminopurine. ThioGDP has previously been shown to bind to EF-Tu (Wittinghofer et al., 1977), and 2-azidoinosine 5’-diphosphate has been shown to bind to the eukaryotic elongation factor 1 (Jerome & Heintz, 1980). The purpose of the work reported here is 2-fold. First, differences in the nucleotide binding site of EF-Tu bound to
-
’
Abbreviations used: EF-Tu, elongation factor Tu; EF-Tuf, nucleotide-free EF-Tu; EF-Ts, elongation factor Ts; thioGTP, 9-O-ribofuranosyl-2-amino-6-mercaptopurine5’-triphosphate; thioGDP, 9-8ribofuranosyl-2-amino-6-mercaptopurine5’-diphosphate; butyl-PBD, 5-(4-biphenylyl)-2-(4-rert-butylphenyl)-l,3,4-oxadiazole;HPLC, performance liquid chromatography.
0006-296018 110420-6265%01.25/0 0 1981 American Chemical Society
6266 B I oc H E M I S T R Y
ECCLESTON
I
::I*
?-----CPM
(b)
\
[Tu GDP] [Tu IhmGDP]
2: Displacement of [3H]GDP from EF-Tu.[~H]GDPby thioGDP. The solutions (total volume 0.26 mL) contained 0.13 rM EF-TwGDP, 7.7 gM [3H]GDP(93 cpm/pmol), 50 pM to 1 mM thioGDP, 7.7 mM Tris-HCI, 3.8 mM MgC12,6.1 mM KCI, 6.1 mM “&I, and 0.7 mM DTT, pH 7.6. After incubation at 20 OC for 10 min, the amount of EF-Tu.[~HH]GDP was determined by cellulose nitrate filtration as described under Materials and Methods. FIGURE
Wavelength (nm)
Structures of the thione and monoanionic forms of thioguanosine and their absorption spectra. Spectra of thioguanosine were recorded in 0.1 M acetate, pH 4.5 (thione form, 84,- = 342 nm), or in 10 mM NaOH (monoanionic form, A, = 320 nm). FIGURE 1:
nucleoside 5’-diphosphate and 5’-triphosphate are determined. Second, the potential of the analogues for allowing the measurement of the equilibrium and kinetic constants of the interactions of EF-Tu in the elongation cycle of protein biosynthesis is investigated. These measurements in the past have been made by using filtration techniques and the ability of EF-Tu to bind to cellulose nitrate. While major advances have been made in understanding the role of EF-Tu in protein biosynthesis by using this technique, it is necessarily rather limited in that measurements are made under nonequilibrium conditions, and fast processes cannot be followed. Since the in vivo rate of peptide elongation is on the order of 20 s-l (Gausing, 1972), the elementary processes of this elongation cycle must be at least as fast as this, and therefore, it is necessary to be able to follow reactions on this time scale. Materials and Methods Assays of EF-Tu-GDP, EF-Tuf, and EF-Ts were performed as described by Miller & Weissbach (1974). EF-TwGDP was prepared from E . coli (Grain Processing Inc., Muscatine, IA) by the method of Leberman et al. (1980) and had a binding capacity of greater than 19 000 pmol of GDP bound per mg of protein. The GTPase activity of the preparations was determined by incubating a solution containing 5 pM EF-Tu. GDP, 100 pM [y3*P]GTP,50 mM Tris-HC1, pH 7.2,50 mM KCl, 50 mM NH,Cl, and 5 mM MgC12 for 4 h at 0 OC or 1 h at 20 or 37 OC. [32P]Piwas then measured after extraction as the phosphomolybdate complex into isobutyl alcoholbenzene (1:l) (Martin & Doty, 1948). In most preparations, no detectable hydrolysis (
