Chapter 7
2+
Interaction of the Vanadyl (VO ) Cation with Guanosine Nucleotides and Elongation Factor Tu 1
1
1,2
1
Anindya Banerjee , Shan Chen , Heike Ruetthard , Fashuh Jiang , Victor W. Huang , Mathias Sprinzl , and Marvin W. Makinen Downloaded by COLUMBIA UNIV on February 4, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch007
1
2
1
1
Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637 Laboratory of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany
2
2+
2+
The vanadyl (VO ) cation is used as a paramagnetic substitute of Mg for electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies to investigate metal-nucleotide and metal-protein interactions in guanosine nucleotide complexes formed with elongation factor Tu (EF-Tu) of Thermus thermophilus. Scatchard plot analysis of the affinity of GDP and GTP to the protein in the presence of VO together with EPR studies showed formation of ternary EF-Tu : VO : guanosine nucleotide complexes of 1:1:1 stoichiometry. While the binding of GDP in the presence of VO was less tight by one order of magnitude than in the presence of Mg , GTP was bound with comparable affinity. By EPR no secondary binding sites on the protein could be detected. Different classes of hydrogens in the active site in the vicinity of the metal ion corresponding to solvent exchangeable and covalently bound hydrogens could be identified by ENDOR for the protein complexes formed in H O or with the recombinant protein purifiedfromE. coli grown in deuteriated minimal medium. VO was also found to support hydrolysis of GTP catalyzed by EF-Tu. These preliminary results show that VO may serve as a sensitive ENDOR probe to investigate active site structure and structural flexibility of residues controlling the conformational change of the protein during GTP hydrolysis. 2+
2+
2+
2+
2
2
2+
2+
During protein biosynthesis in bacteria, the elongation factor Tu (EF-Tu) recognizes, transports, and positions the codon-specified aminoacyl-transfer RNA onto the A site of the ribosome (7). In its cellular role, the interactions of EF-Tu with other factors important in protein biosynthesis are governed by the binding of GTP and GDP, which act as effector molecules to control the conformation of the protein. Not only is there a marked change in the affinity of binding of these guanosine nucleotides by two orders of magnitude in the presence of Mg as a required divalent metal ion cofactor 2+
104
©1998 American Chemical Society In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
105 (2, 5), but also, as shown for the proteinfromThermus thermophilus, EF-Tu exhibits a dramatic conformational change in the nucleotide binding region upon GTP hydrolysis (4-7). In the triphosphate complex, residues 54-59 (T. thermophilus numbering) form a small helix which assume an extended conformation in the diphosphate complex. While Asp-51 is coordinated to the metal ion in both diphosphate and triphosphate complexes, Thr-25 and Thr-62 supply hydroxyl groups to coordinate the divalent metal ion together with the terminal phosphate group of GTP in the trinucleotide complex. Upon nucleotide hydrolysis, however, solvent molecules replace the yphosphate group, and the side chain of Thr-62 moves to a new position 16 A distant from its site in the ternary EF-Tu : Mg : GTP complex. The influence of the metal ion and how changes in metal-ligand interactions control the conformation of the protein in the effector binding region are not known. To investigate the influence of metal-ligand interactions on the dynamics of protein structural changes requires a spectroscopic approach. Unfortunately, Mg , as the naturally occurring cofactor, is spectroscopically silent. The vanadyl (V0 ) cation is a versatile paramagnetic substitute for divalent metal ions in macromolecules (8\ and we have found that it not only substitutes specifically for Mg , but it also mimics closely the interactions of Mg with nucleotides (9-12). By employing V 0 as a paramagnetic probe for electron nuclear double resonance (ENDOR) spectroscopy, we have determined the detailed structure of V0 -nucleotide complexes in solution showing that they are identical to Mg complexes defined by X-ray crystallography. In these studies the precision of structure analysis in the form of spectroscopically determined metal-nucleus distances over a 38 A range was at a level of accuracy exceeded only by that of X-ray diffraction. We have consequently turned our attention to the use of V 0 as a paramagnetic probe of metal coordination geometry and active site structure in EF-Tu. In this report we present preliminary results showing that the binding of V 0 to EF-Tu is specific and competitive with Mg and in the presence of GDP and GTP it forms ternary EF-Tu : V 0 : guanosine nucleotide complexes of 1:1:1 stoichiometry.
Downloaded by COLUMBIA UNIV on February 4, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch007
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
Experimental Procedures General. All guanosine nucleotides were obtained as their sodium saltsfromSigma (St. Louis, MO 63178); vanadyl sulfate hydrate, deuterium oxide (> 99%), and pmercaptoethanol from Aldrich (Milwaukee, WI 53233); urea (ultrapure, > 99.9%) from Amresco (Solon, OH 44139); aqueous solutions of [8- H]GDP and [8- H]GTP as ammonium salts with a specific activity of 11.5 Ci/mmol and a radioactive concentration of 1.0 mCi/mL were obtainedfromAmersham Life Sciences (Arlington Heights, IL 60005). [y- P]GTP was obtained from Hartmann Analytik (Braunschweig, Germany). Nitrocellulose membranes (HA 0.54 |im) were purchased from Millipore Corporation (Belford, MA 01730). Filter-Count scintillationfluidfrom Packard Instruments (Meriden, CT 06450) was used to detect radioactivity in a Packard Instruments MinaxP Tri-Carb 4000 Series Liquid Counter. Chelex resin (100-200 mesh biotechnology grade) was obtained from Bio-Rad Laboratories (Hercules, CA 94547). Bovine serum albumin (BSA) was obtainedfromBoehringer Mannheim (Indianapolis, IN 46250); DNase I (from bovine pancreas) and lyzozyme (from chicken egg white), Q Sepharose Fast Flow (anion exchanger), and CM 3
32
In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
3
106
Downloaded by COLUMBIA UNIV on February 4, 2015 | http://pubs.acs.org Publication Date: December 10, 1998 | doi: 10.1021/bk-1998-0711.ch007
Sepharose CL-6B (cation exchanger) were obtained from Sigma. Sephacryl S-200 H R (gel filtration medium) and column systems C 16/20, X K 26/60 and X K 26/100 were obtained from Pharmacia Biotech, Inc. (Piscataway, N J 08854). Purification of EF-Tu. Cell paste of Escherichia coli (JM109) engineered (73) with the tufl gene of Thermus thermophilus (HB8) was provided by Philip Johnson of the Pilot Plant, Department of Biochemistry, University of Wisconsin (Madison, WI 53705). Thermostable EF-Tu was isolated and purified from cell paste using the procedure of Blank et al (14). Variations in the procedure were as follows: Cell lysis was performed by freeze-thaw cycles instead of nitrogen decompression. In each cycle the suspension was frozen in a methanol-dry ice bath (-80° C) and then allowed to thaw to 0° C. This cycle was repeated four times. All subsequent procedures were carried out at 4° C. The homogenate obtained was centrifuged at 8000 x g for 90 min to remove cell debris. The supernatant was then centrifuged at 113,000 x g for 3 hr, and to the clear supernatant (150 mL) solid (NFL^SC^ was added under gentle stirring to 70 % saturation over a 2-3 hr period. After the addition of (NFL^SC^, the mixture was stirred for another hour and then allowed to stand overnight at 4° C. The protein was dissolved in and dialyzed against the anion exchange-buffer (14) and applied to a 2 x 10 cm Q Sepharose Fast-Flow column. The fractions containing the protein were concentrated to a volume of 5 mL by ultrafiltration using a membrane with minimum molecular weight cut-off of 10,000 (Amicon, Inc., Beverly, M A 01915). The protein was stored in 50 % (v/v) glycerol at -20° C for further use. Nucleotide free EF-Tu was obtained based on procedures described by Limmer et al. (15). The fractions containing the protein, eluted from the cation exchange column, were pooled and dialyzed against 0.2 M NaCl (5 x 100 mL), buffered to pH 7.5 with 0.025 M PIPES, and concentrated to a final volume of 20 mL using Centriprep-10 Concentrators (Amicon). Protein concentration was determined using the bicinchoninic acid assay (Sigma), based on the method of Smith et al. (16). Perdeuteriated EF-Tu was obtained by growth of E. coli (JM109) cells (73) on minimal medium containing H20 and [ H ]acetate (6 g/L) in addition to M 9 salts and vitamins. Perdeuteriated EF-Tu was then obtained by treatment of cells as grown under natural abundance isotope conditions. The purified EF-Tu was found to be enriched to > 98% deuteriation by MALDI-TOF mass spectrometry. 2
2
3
Measurement of Nucleotide Binding Affinity of EF-Tu. The interaction of guanosine nucleotides with EF-Tu was measured by application of the procedure of Arai and coworkers (2, 3, 17). The incubation mixture contained 0.05 M KC1 buffered to pH 7.5 with 0.05 M HEPES. The concentration of EF-Tu was 8.8 x 10" M and the guanosine nucleotide concentration was varied from 12.5 to 500 x 10* M in a constant reaction volume of 0.175 mL. After an aliquot of the incubation mixture was applied to the nitrocellulose membrane, the filter was washed with three aliquots of 0.05 M KC1 buffered to pH 7.5 with 0.05 M HEPES. Binding of radioactive GDP or GTP to the membrane independent of added protein was also detected, particularly at high nucleotide concentrations. The observed counts for binding in the presence of EF-Tu were corrected for this background binding activity. 9
9
In Vanadium Compounds; Tracey, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.
107
To measure the binding affinity of nucleotides to EF-Tu in the presence of M n a n d V 0 a s w e l l as in the absence of added metal ion, the protein and nucleotide were treated first with a suspension of Chelex to ensure removal of all trace divalent metal ions, including low levels of M g (assessed to be
