Proc. Natl. Acad. Sci. USA Vol. 91, pp. 9828-9831, October 1994 Biochemistry Mechanism of GTP hydrolysis by G- a subunits CHRISTIANE KLEUSS*, ANDRE S. RAW*, ETHAN LEE*, STEPHEN R. SPRANGt, AND ALFRED G. GILMAN* *Department of Pharmacology and tHoward Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Contributed by Alfred G. Gilman, July 12, 1994

ABSTRACT Hydrolysis of GTP by a variety of the arginine residue that is ADP-ribosylated by -binding is a crucial step for regulation of in Gsa (Arg-178 in Gial; Arg-201 in GsL) (18). these biological switches. Mutations that impair the GTPase Noel et al. (19) have recently solved the structure of Gta, activity ofcertain heterotrimeric signal-transducing G proteins and Coleman et al. (32) have determined the structure ofGi.j. or ofp21m cause tumors in man. A conserved glutamic residue The structures of the guanosine 5'-[y'.thio]triphosphate in the a subunit of G proteins has been hypothesized to serve (GTPyS)-bound forms of these proteins [and of the Gln-204 as a general base, thereby activating a water molecule for -- Leu (Q204L) mutant of Giad] again offer little insight into nucleophilic attack on GTP. The results of mutagenesis of this the role of the critical glutamine residue. However, Noel et residue (Glu-207) in Gial refute this hypothesis. Based on the al. (19) speculated that a glutamic residue (Glu-204 in Gta; structure of the complex of Gial with GDP, Mg2+, and AMFZ, Glu-207 in Giai) that is conserved in G-protein a subunits but which appears to resemble the transition state for GTP hydro- corresponds to Tyr-64 in p2lm could rotate so that its lysis, we believe that GIn-204 of G1.1, rather than Glu-207, carboxylate side chain might deprotonate or polarize a crys- supports of GTP hydrolysis by stabilization of the tallographically observed water molecule positioned for in- transition state. line attack on the y- group of GTP. We have mutated Glu-207 in Giai to glutamine or alanine and charac- Heterotrimeric G proteins play important roles as transduc- terized the altered proteins in an attempt to confirm or refute ers and timers in a variety ofintracellular the model of GTP hydrolysis proposed by Noel et al. systems (1-5). G proteins are activated by binding of GTP in exchange for GDP. This reaction is catalyzed by interaction of the inactive, GDP-bound with an appropriate MATERIALS AND METHODS agonist-bound receptor. Binding of GTP to the G protein Plasmids and Proteins. A cDNA containing the entire causes conformational changes that result in dissociation of coding sequence of Gial (20) was ligated into the EcoRI site the GTP-liganded a subunit from both the receptor and a of M13mpl9. Mutagenesis was performed by the method of dimer of the G protein f3 and y subunits. The activated a Kunkel et al. (21), using 5'-CCACTTCTTCCGCTGC- subunit and the fry dimer can then interact with and regulate GATCGTTGGCCTCCCACG-3' as the mutagenic oligonu- the activities of downstream effectors, such as adenylyl cleotide to generate E207Q Giai and 5'-CCACTTCTTC- , phospholipases, and channels. CGCGCAGATCTCTGGCC-3' to generate E207A Giai. The G proteins deactivate themselves as a result of their mutations were confirmed by DNA sequencing. The com- intrinsic GTPase activity. The kcat for this reaction is very plete coding sequences (Nco I-EcoRI fragments) of the two slow, typically about 5 min- for most members ofthe G, and mutants and the wild-type protein were subcloned into the Gi subfamilies of G proteins (6-8). However, in some cases Nco I and Sma I sites of plasmid H6pQE60 (22) to generate G protein-coupled effectors or other molecules can act as cDNAs encoding proteins with hexahistidine sequences at GTPase-activating proteins, increasing this rate to -50 min1 their amino termini. These plasmids were transformed to- or greater (9). Mutations that impair the GTPase activity of gether with plasmid pREP4 into Escherichia coli BL21(DE3). Gsa and Gia proteins have been associated with endocrine Proteins were expressed and purified on Ni2+-NTA resin as tumors, particularly ofthe pituitary (10). ADP-ribosylation of the were confirmed by protein an arginine residue in Gsa by cholera toxin also inhibits described (22); mutations GTPase activity (11). The result of this modification is sequencing. Mutants R178C Gi,, and Q204L Gial (not histi- irreversible activation of the G-protein a subunit with cor- dine-tagged) were generated, expressed, and purified as responding stimulation of its effector, adenylyl . described (22, 23). The sequences of these mutants were Detailed understanding of the mechanism of GTP hydro- confirmed by DNA sequencing, and their crystal structures lysis by guanine nucleotide-binding proteins was, until re- have been solved. cently, largely based on studies of p2lr" and Trypsinization of Giai. Wild-type or mutant Gja1 protein (1 EF-Tu, for which high-resolution crystal mg/ml) was incubated for 150 min at 300C in 50 mM Na- structures are available (12-16). The guanine nucleotide- Hepes, pH 8.0/1 mM EDTA/5 mM dithiothreitol/10 mM binding domains of these proteins are highly homologous to MgSO4 plus either 100 1LM concentrations of GDP, GTP, or those of G-protein a subunits. Particularly vexing has been GTPyS or 30 pLM AlCl3 and 10 mM NaF. Trypsin was added the role ofGln-61 in p2lms. Although mutation ofthis residue (26 pg/ml) as indicated. After 10 min at 300C, sample buffer drastically inhibits GTPase activity and is a common cause of was added and samples were boiled. human malignancies, elucidation ofthe structure ofwild-type GTP'yS Binding Kinetics. Proteins (10 pg/ml) were incu- and mutant p21ras proteins has not fully defined the role ofthis bated at 300C in 100 mM NaHepes, pH 8.0/1 mM EDTA/10 important residue. Mutations ofthe corresponding glutamine mM dithiothreitol/10 mM MgSO4/2 pM [35S]GTPyS (700 residue in heterotrimeric G proteins (Gln-204 in Gial; Ghn-227 cpm/pmol). Aliquots (100 jud) were withdrawn at the indi- in GSGL) also impair GTPase activity (17), as do mutations of cated times, added to 2 ml of ice-cold wash buffer (20 mM Tris HCl, pH 8.0/100 mM NaCl/25 mM MgCl2), and ana- lyzed as described (24). Values ofkapp for the binding reaction The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: GTPyS, guanosine 5'-[Lythio]triphosphate. 9828 Downloaded by guest on September 25, 2021 Biochemistry: Kleuss et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9829 were calculated by a nonlinear least-squares fit to the equa- that of Q204L Gia1, since the amount of protein protected tion B = Bq(1 - e-kt). after incubation with GTP is lowerfor the R178C mutant (Fig. Steady-State Hydrolysis of GTP. Proteins (120 nM) were 1, lane 18). incubated at 300C in 50mM NaHepes, pH 8.0/1 mM EDTA/5 A1FZ and Mg2+ bind to the GDP-bound form of G-protein mM dithiothreitol/10 mM MgSO4/5 puM [y32P]GTP (3200 a subunits and cause many of the changes seen with nonhy- cpm/pmol). Aliquots (50 A4) were removed at the indicated drolyzable analogs of GTP, including protection from tryptic times and [32p]p; was determined as described (6). proteolysis (Fig. 1, lane 5) (27). It is generally believed that Steady-State Binding of GTPyS and GTP. Proteins (150 the A1FZ complex mimics the -phosphate of GTP and thus nM) were incubated for 3 hr at 300C in 100 mM NaHepes, pH activates G-protein a subunits in concert with GDP (28, 29). 8.0/1 mM EDTA/10 mM dithiothreitol/10 mM MgSO4 with Of interest, Q204L Giai is completely degraded by trypsin in 2 pM [35S]GTPyS (700 cpm/pmol) or 2 pM [y-32P]GTP (3300 the presence of AlFZ, suggesting that AIF4 does not bind to cpm/pmol). The reaction was quenched by addition of ice- or activate the mutant protein. The crystal structure of the cold wash buffer and analyzed as described (24). complex of A1F- with GDP-Giaj reveals interactions be- Determination of k for Hydrolysis of GTP. Proteins (425 tween Gln-204 and the AlFZ ligand that are consistent with nM) were incubated for 15 min at 30°C and then for 5 min at these possibilities (see below). 20°C in 50 mM NaHepes, pH 8.0/10 mM EDTA/5 mM Guanine Nucleotide Binding and Hydrolysis. All of the dithiothreitol/2 ,uM [y-32P]GTP (7300 cpm/pmol). Reactions proteins used in these studies bound GTPyS to a similar (at 20°C) were started by addition of MgSO4 and GTPyS to extent at equilibrium (usually about 0.7 mol/mol ofprotein). final concentrations of 20 mM and 200 uM, respectively. The rate of binding of GTP-yS to these proteins is limited by Aliquots (50 j4) were analyzed for [32p]P; at the indicated the rate of dissociation of GDP; this value for wild-type Giai times as described (6). (kap = 0.027 min-') was essentially identical to that reported earlierfor the non-histidine-tagged protein (7) and to the rates determined for the E207Q and E207A mutants (Fig. 2). RESULTS Similar rates ofGTPVS binding were also observed for R178C Protein Purification. Wild-type Gial and two mutants Giai (Fig. 2) and for the Q204L mutant (data not shown). The (E207Q and E207A) were expressed with six histidine resi- rates of dissociation of GTPyS from all five proteins were dues preceding the initiator methionine of Giai and were exceedingly slow (data not shown). purified in one step using Ni2+-NTA agarose. The Q204L and The steady-state rate of GTP hydrolysis by the five pro- R178C mutants of Giai did not contain amino-terminal histi- teins is shown in Fig. 3. This rate is equal to the rate ofGTPyS dine residues and were purified conventionally. In each case binding (and GDP dissociation) for the wild type and Glu-207 =40 mg ofhighly purified protein was obtained from a 1-liter mutants, while it is substantially reduced for R178C and bacterial culture (Fig. 1, lanes 1, 6, 11, 16, and 21). Addition Q204L Giai. Thus, steady-state GTP hydrolysis is limited by of histidine residues at the amino terminus does not alter the GDP dissociation for the wild-type protein and the Glu-207 kinetics ofguanine nucleotide binding or hydrolysis by Giai. mutants, while the kcat for GTP hydrolysis by R178C or Trypsinization. Certain conformational changes that ac- Q204L Giai is slower than the rate ofproduct dissociation and company activation of G-protein a subunits by GTP are substrate binding. detectable by enhancement of intrinsic tryptophan fluores- The fractional steady-state occupancy of the guanine nu- cence (25) or acquisition of resistance to proteolysis by cleotide- of a G-protein a subunit by GTP (com- trypsin (26). GDP-bound a subunits are readily hydrolyzed pared with GTPyS) is given by the expression F(GTP) = by trypsin to relatively small fragments; GTP- or GTPyS- kdiss,GDP/(kcat,GTP + kdiss,GDP). Thus, if kcat is high relative to activated forms of these polypeptides are rapidly cleaved kdiss,GDP, the G protein exists largely in the GDP-bound form near the amino terminus, and 37- to 39-kDa products then at steady state. Binding of[y_32PJGTP to the five proteins was accumulate. When wild-type G-protein a subunits are incu- compared with that of [355]GTP'yS after a 3-hr incubation bated with GTP, the GDP-bound form of the protein pre- (Fig. 4). Fractional occupancies by [y_32PJGTP for E207Q dominates because the rate of hydrolysis of GTP greatly Giai and E207A Giai (0.012% and 0.014%) were only slightly exceeds the rate of release of product (GDP). Wild-type Gi., greater than the value for the wild-type protein (none detect- and the E207Q and E207A mutants are protected from ed), indicating an insignificant difference in ka values among proteolysis after incubation with GTPyS but not with GTP, these three proteins. The high fractional occupancies by GTP indicating the likelihood that these proteins all have signifi- for R178C and Q204L Gial are consistent with previous cant GTPase activity (Fig. 1). In contrast, the GTPase- estimates ofthe effects ofthese mutations in Gsa (17, 18). The deficient proteins R178C Gi., and Q204L Giai are protected value of kat for R178C Gial calculated from the data of Fig. following incubation with either GTPyS or GTP. R178C Giai 4 is 0.014 min-1, which is >100-fold lower than the value appears to have a GTPase activity that is slightly higher than observed for wild-type Gial (Fig. 5 and ref. 7). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2, 22 23 24 25

GDP GTP -. _ - - GTPyS + AMF - -+ - - - +-. + + + + + t trypsin + -s

FIG. 1. Trypsinization of wild-type and mutant forms of Giaj. Wild-type or mutant Giai was incubated with either GDP, GTP, GTP-S, or AlCl3/MgCI2/NaF (AMF) and trypsin where indicated. Proteins were separated in a sodium dodecyl sulfate/11% polyacrylamide gel and stained with Coomassie blue. Lanes 1-5, wild-type Giai; lanes 6-10, E207A Giai; lanes 11-15, E207Q Giai; lanes 16-20, R178C Gia1; lanes 21-25, Q204L Giai. Downloaded by guest on September 25, 2021 9830 Biochemistry: Kleuss et al. Proc. Natl. Acad. Sci. USA 91 (1994)

Li2 El (i

100 l-] E E

17 Ex E 75 -[I , 0 s0 W- 0- 0

'a 50 - c w co0~ 0. C.) I9- 0 C!, 'E 25 r_

U

0 30 60 90 120 150 180 0- Time (min) WT E207A E207Q R1 78C 0204L 0 00 1C i1 41 00 100.00 c kinetics. Proteins were treated as FIG. 2. [35S]GTPyS binding FIG. 4. Steady-state binding of [v-32P]GTP and [35S]GTP'yS. described in Materials andMethods. Data are normalized to binding occupancy is expressed as binding of [y-32P]GTP as a after 180 min. Maximal binding according to amount of protein was Fractional 58% for wild-type (WT) Gij, 76%6 for E207A Gial, 70%6 for E207Q percentage of that observed for [35S]GTPyS. 65% for R178C Giai, and 70%6 for Q204L Gial. Values of kapp Gial, charge developing on the water molecule in were calculated by a nonlinear least-squares fit to the equation B = stabilize positive state. If any of these mechanisms was opera- Beq(l - e-k). the transition tive, mutation ofGlu-207 to glutamine should slow the rate of Values for kt can be calculated directly by observation of GTP hydrolysis and mutation to alanine should have a a single round of GTP hydrolysis. This is accomplished (Fig. substantial deleterious effect. The biochemical data reported 5) by allowing the protein to bind GTP in the absence of here are not consistent with a role for Glu-207 in GTP Mg2+. Catalysis is then initiated by addition of Mg2+, and hydrolysis, leaving moot the question of the conservation of rebinding of radiolabeled substrate is prevented by addition this residue among all G-protein a subunits. The correspond- ofunlabeled guanine nucleotide. These experiments indicate ing residue in p2lm, Tyr-64, does appear to be necessary for kcat values of 3.4 min-', 1.1 min-1, and 2.2 min-' for GTPase activity, although hydrophobic substitutions are wild-type Gial, E207A Gi.1, and E207Q Gi.1, respectively. tolerated (30). R178C Gi., and Q204L Giai did not hydrolyze detectable Coleman et al. (32) have recently solved the structure ofthe amounts ofGTP over the short time frame ofthis experiment. complex of Gia1 with GTPyS. In this structure (and that of GTPvS-Gt,), Gln-204 is close to the putative nucleophilic water molecule. A small rotation would place the carboxa- DISCUSSION mide side chain adjacent to the water. The structure ofQ204L A water molecule is visualized in the crystal structures ofGta GiaQ has also been solved and reveals no significant pertur- and Giai (as well as in p2lr and EF-Tu) that is ideally bations, suggesting that the explanation for the impaired positioned for in-line nucleophilic attack on the -y-phosphate GTPase activity of this mutant lies with the chemistry of the group ofGTP. Noel et al. (19) have suggested that rotation of side-chain substitution rather than with any conformational the side chain of Glu-207 would bring the carboxylate group or stearic effects. Although mutation of Gln-61 in p21r to of this residue into position to act as a general base, depro- glutamic acid was originally thought to impair GTPase ac- tonating and activating the water molecule. Alternatively, the carboxylate group could polarize the water molecule or

Tumover nuimber 1.0 - oWT 0.02!6/min-1 C E207A 0.02!6/min1 ._ .C 0.8- 0 E207Q 0.02 0O0 CD A R178C 0.0C )6/min-1 v Q204L 0.OC )6/min-1 O 0.6- E A- 0.02 C0.0 X 0.4- E .5 CL0.01 E X 0.2

0.0 It 20.0 21.0 22.0 23.0 0 10 20 30 Time (min) Time (min) FIG. 5. Determination of kat for the hydrolysis of GTP. After 21 FIG. 3. Steady-state hydrolysis of [e-32P]GTP. Proteins were min, MgSO4 and GTP'yS were added to final concentrations of20 mM treated as described in Materials and Methods. Aliquots (50 A4) were and 200 pM, respectively. Aliquots (50 p4) were analyzed for [32P]P1 removed at the time points indicated and [32p]pi was determined. at the indicated times. Downloaded by guest on September 25, 2021 Biochemistry: Kleuss et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9831 tivity, it is now known that the kt for this mutant is increased 9. Berstein, G., Blank, J. L., Jhon, D. Y., Exton, J. H., Rhee, 20-fold, although its Km for GTP is, not surprisingly, much S. G. & Ross, E. M. (1992) 70, 411-418. higher (31). Thus, this observation is also consistent with the 10. Lyons, J., Landis, C. A., Harsh, G., Vallar, L., Grunewald, a role in catalysis. This K., Feichtinger, H., Duh, Q.-Y., Clark, 0. H., Kawasaki, E., view that Gln-61 plays distinct Bourne, H. R. & McCormick, F. (1990) Science 249, 655-659. mutation demonstrates that placement ofa base at position 61 11. Cassel, D. & Selinger, Z. (1977) Proc. Natd. Acad. Sci. USA 74, can accelerate catalysis, presumably by activation or stabi- 3307-3311. lization of the nucleophilic water molecule, but it does not 12. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J. prove that this is the role normally played by glutamine at this & Wittinghofer, A. (1989) Nature (London) 341, 209-214. position. Finally, Coleman et al. (32) have determined the 13. Brunger, A. T., Milburn, M. V., Tong, L., deVos, A. M., structure of Gia1 complexed with GDP, AIFZ, and Mg2+. Jancarik, J., Yamaizumi, Z., Nishimura, S., Ohtsuka, E. & the assumes a six-coordinate octahedral coordi- Kim, S.-H. (1990) Proc. Natd. Acad. Sci. USA 87, 4849-4853. Here, A13+ 14. Jurnak, F. (1985) Science 230, 32-36. nation sphere with its four fluorine atoms lying in one plane; 15. Berchtold, H., Reshetnikova, L., Reiser, C. 0. A., Schirmer, the f-phosphate oxygen atom and the presumptive nucleo- N. K., Sprinzl, M. & Hilgenfeld, R. (1993) Nature (London) philic water molecule lie trans to each other. Thus, this 365, 126-132. complex appears to mimic a structure approaching the tran- 16. Kjeldgaard, M., Nissen, P., Thirup, S. & Nyborg, J. (1993) sition state for GTP hydrolysis. Significantly, Gln-204 has Structure 1, 35-50. moved from its ground-state position in the GTPyS structure 17. Graziano, M. P. & Gilman, A. G. (1989) J. Biol. Chem. 264, to a position in which its carboxamide side chain is within 15475-15482. 18. Freissmuth, M. & Gilman, A. G. (1989) J. Biol. Chem. 264, hydrogen-bonding distance of both the nucleophilic water 21907-21914. molecule and a fluorine atom. Glu-207 has not moved in this 19. Noel, J. P., Hamm, H. E. & Sigler, P. B. (1993) Nature (Lon- structure. We thus support the model (for both G-protein a don) 366, 654-663. subunits and p21s) where Gln-204 (Giai) or Gln-61 (p2lm) 20. Jones, D. T. & Reed, R. R. (1987) J. Biol. Chem. 262, 14241- supports catalysis of GTP hydrolysis by stabilization of the 14249. transition state. This may include polarization and orienta- 21. Kunkel, T. A., Roberts, J. D. & Zabour, R. A. (1987) Methods tion of the attacking water molecule and/or stabilization of Enzymol. 154, 367-382. 22. Lee, E., Linder, M. E. & Gilman, A. G. (1994) Methods developing charge in the transition-state complex. Enzymol. 237, 146-164. 23. Coleman, D. E., Lee, E., Mixon, M. B., Linder, M. E., Berg- We thank Pamela Sternweis for excellent technical assistance. huis, A. M., Gilman, A. G. & Sprang, S. R. (1994) J. Mo!. Biol. This work was supported by American Cancer Society Grant 238, 630-634. BE3O0-, National Institutes of Health Grant GM34497, Welch 24. Northup, J. K., Smigel, M. D. & Gilman, A. G. (1982) J. Biol. FoundationGrant1-1271, afellowshipfromtheDeutscheForschungs- Chem. 257, 11416-11423. gemeinschaft to C.K., The Lucille P. Markey Charitable Trust, and 25. Higashijima, T., Ferguson, K. M., Sternweis, P. C., Ross, The Raymond Willie Chair of Molecular Neuropharmacology. E. M., Smigel, M. D. & Gilman, A. G. (1987) J. Biol. Chem. 262, 752-756. 1. Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649. 26. Fung, B. K.-K. & Nash, C. R. (1983) J. Biol. Chem. 258, 2. Simon, M. I., Strathmann, M. P. & Gautam, N. (1991) Science 10503-10510. 252, 802-808. 27. Sternweis, P. C. & Gilman, A. G. (1982) Proc. Nat!. Acad. Sci. 3. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M. & Satoh, T. USA 79, 4888-4891. (1991) Annu. Rev. Biochem. 60, 349-400. 28. Bigay, J., Deterre, P., Pfister, C. & Chabre, M. (1985) FEBS 4. Bourne, H. R., Sanders, D. A. & McCormick, F. (1991) Nature Lett. 191, 181-185. (London) 349, 117-127. 29. Higashijima, T., Graziano, M. P., Suga, H., Kainosho, M. & 5. Hepler, J. R. & Gilman, A. G. (1992) Trends Biochem. Sci. 17, Gilman, A. G. (1991) J. Biol. Chem. 266, 33%-3401. 383-387. 30. Nur-E-Kamal, M. S. A., Sizeland, A., D'Abaco, G. & Maruta, 6. Higashijima, T., Ferguson, K. M., Smigel, M. D. & Gilman, H. (1992) J. Biol. Chem. 267, 1415-1418. A. G. (1987) J. Biol. Chem. 262, 757-761. 31. Frech, M., Darden, T. A., Pedersen, L. G., Foley, C. K., 7. Linder, M. E., Ewald, D. A., Miller, R. J. & Gilman, A. G. Charifson, P. S., Anderson, M. W. & Wittinghofer, A. (1994) (1990) J. Biol. Chem. 264, 8243-8251. Biochemistry 33, 3237-3244. 8. Graziano, M. P., Freissmuth, M. & Gilman, A. G. (1989) J. 32. Coleman, D. E., Berghuis, E. L., Linder, M. E., Gilman, Biol. Chem. 264, 409-418. A. G. & Sprang, S. R. (1994) Science, in press. Downloaded by guest on September 25, 2021