COMMUNICATION
Structure of Gialpha 1·GppNHp, Autoinhibition in a Galpha Protein-Substrate Complex*

David E. ColemanDagger and Stephen R. SprangDagger §

From the § Howard Hughes Medical Institute and the Dagger  Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The structure of the G protein Gialpha 1 complexed with the nonhydrolyzable GTP analog guanosine-5'-(beta gamma -imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 Å. In the active site of Gialpha 1·GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the gamma -phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5'-O-3-thiotriphosphate or the transition state analog GDP·AlF4-. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein alpha  subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Galpha .

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Galpha subunits of heterotrimeric G proteins are GTP hydrolyases which, upon receptor activation, bind to GTP and regulate effector molecules (1, 2). Galpha -catalyzed hydrolysis of GTP to GDP releases the Galpha ·GDP product complex from effector and allows its sequestration by inhibitory Gbeta gamma subunits. The rate of the hydrolytic reaction thus determines, in part, the length of time during which the effector is regulated by activated G proteins.

Structures of the Galpha subunits Gtalpha (transducin), Gialpha 1, and Gsalpha complexed with Mg2+ and the nonhydrolyzable GTP analog GTPgamma S1 have provided views of the active site in the ground state (7-9). In particular, the positions of the presumptive nucleophilic water (Wnuc), Mg2+, and the side chains of catalytically significant residues have been determined. Structures of Gialpha 1 and Gtalpha complexed with GDP·AlF4-·Wnuc, a mimic of the pentavalent transition state for GTP hydrolysis, have also been determined (8, 10).

Mutagenesis experiments demonstrate that two residues of Gialpha 1, Arg178 and Gln204, are required for catalysis (11, 12). In crystals of Gialpha 1·GTPgamma S, which mimics the ground state E·S complex, the side chains of these residues are partly disordered and do not interact with the substrate analog. In contrast, in complexes of Gialpha 1 and its homolog Gtalpha with GDP·AlF4-, both residues are well ordered and interact with AlF4- and its axial water ligand (8, 10). It was proposed that Arg178 and Gln204 stabilize the pentavalent transition state through an analogous set of interactions (8, 10). It was also suggested that the low catalytic rate (kcat approx  2-4 min-1) exhibited by Gialpha 1 (14) and its homologs might be attributed to a high activation energy for the conformational rearrangement of Arg178 and Gln204 from the ground state to the transition state (2). However, the nature of this rearrangement is not established, in part because the Gialpha 1·GTPgamma S complex may not accurately mimic the true E·S complex. Specifically, the thiol substituent of the gamma -phosphate in GTPgamma S may sterically perturb the catalytic site. The sulfur atom is both more bulky than the corresponding oxygen atom of GTP (van der Waals radii of 1.8 and 1.4 Å, respectively) and has a longer bond length with the gamma -phosphate atom (P-S, 1.86 Å; P-O, 1.52 Å). Hence, to obtain a more accurate view of the E·S ground state, we have determined a high resolution x-ray crystal structure of Gialpha 1 complexed with an alternative nonhydrolyzable GTP analog: GppNHp. In GppNHp all three terminal substituents of the gamma -phosphate group are oxygen atoms.

The active site of the Gialpha 1·GppNHp complex differs from that with GTPgamma S in several respects, but most importantly in the conformation of the catalytic residue Gln204. In this conformation, Gln204 interacts with Wnuc, but neither contacts the nucleotide nor is positioned to interact with the pentavalent transition state. Thus, Gln204 may participate directly in the Gialpha 1·GTP ground state but must reorient to stabilize the transition state. RGS4, a member of the RGS family of G protein stimulatory factors (15, 16), may accelerate hydrolysis of GTP by Gialpha 1, in part, by destabilizing the ground-state conformation of Gln204.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Non-myristoylated, recombinant rat Gialpha 1 was synthesized in E. coli and purified as described previously (17). Crystals of Gialpha 1 complexed with GppNHp and Mg2+ were grown and prepared for x-ray data collection as described except that GppNHp was substituted for GTPgamma S (18). Crystals were transferred to a cryoprotection solution containing 15% (v/v) glycerol and frozen in liquid propane as described (19). Two x-ray diffraction data sets were collected at 100 K, each from a single crystal. The first was measured at the Cornell synchrotron source (CHESS) A1 line (lambda  = 0.91 Å) equipped with an ADSC Quantum 1 CCD detector. The crystal diffracted beyond 1.5 Å, but data were measured only to 1.7 Å. A second data set collected to 1.5 Å was collected at the CHESS F1 line (lambda  = 0.92 Å) equipped with an ADSC Quantum 4 CCD detector. Data were processed using the DENZO/SCALEPACK programming package (20). After simultaneous determination of the scale factors for both data sets using all observations, data between 15-2.24 Å from the first data set and between 2.24-1.50 Å from the second were combined to obtain the final 15.00-1.50-Å data set (Table I).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Data collection and final refinement statistics

The structure of the Gialpha 1·GppNHp complex was solved by molecular replacement, using the Gialpha 1·GTPgamma S complex (PDB accession code 1gia) with the nucleotide, Mg2+, and with waters removed as the starting model. SigmaA-weighted Fo - Fc and Fo - Fc difference maps (21) indicated strong difference electron density for GppNHp, a previously unobserved active site water molecule, and new side chain positions for Glu43 and Gln204. Small changes in the side chain conformations of other residues were also observed. Simulated annealing omit maps (22) were used to confirm that the observed differences were not caused by model bias. The protein model was manually rebuilt and ligands were added using the interactive graphics model building program O (23). Positional and atomic temperature factor refinement was carried out on the rebuilt model using XPLOR (24) followed by additional rounds of model building and refinement. In the final rounds, a bulk solvent correction (25) was applied using XPLOR. Electron density about the active site is shown in Fig. 2. The free-R factor, computed using 5% of the data, was used to monitor the refinement (26). The structure exhibits good stereochemistry, with 94% of the residues in the most favored regions of the Ramachandran plot as analyzed by PROCHECK (27). Comparisons and superposition of the model with other proteins was carried out using O. Data collection and refinement statistics are given in Table I.

The model of the RGS4·Gialpha 1·GppNHp complex was made by superimposing the Calpha atoms of residues 34-343 of Gialpha 1 from the Gialpha 1·GppNHp complex onto the corresponding atoms of the RGS4·Gialpha 1·GDP·AlF4- complex (PDB accession code 1agr). Gialpha 1·GDP·AlF4- was then replaced by the superimposed Gialpha 1·GppNHp complex. Analysis and in silico mutations were then performed on the resulting RGS4·Gialpha 1·GppNHp model using O.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The structure of the Gialpha 1·GppNHp complex (Fig. 2A) differs from that of Gialpha 1·GTPgamma S (Fig. 2B) in two ways. First, there are small changes in the side-chain positions of several surface residues that are poorly ordered in crystals of Gialpha 1·GTPgamma S. These differences are most likely because of damping of thermal motions in the frozen Gialpha 1·GppNHp crystals. In contrast, 2.0 Å data from Gialpha 1·GTPgamma S crystals were collected at 14 °C. Additionally, the Gialpha 1·GppNHp data set was measured to 1.5 Å, permitting unambiguous assignment of side chain conformations.

The second, and more interesting, group of changes relative to Gialpha 1·GTPgamma S are observed in the active site and are most likely attributable to GppNHp (Fig. 2A). For the most part, the active site is similar to that observed in the Gialpha 1·GTPgamma S complex (Fig. 2B). All of the interactions between the guanine nucleotide, Mg2+, and the protein observed in the latter complex are also present in Gialpha 1·GppNHp. The presumptive water nucleophile (Wnuc) is also clearly observed (Fig. 1). A new feature of the active site is a highly ordered (B = 21.9Å2) water molecule (W600) bound to the O1G oxygen of the gamma -phosphate (Fig. 1 and Fig. 2A). Modeling indicates that this water molecule cannot bind to the Gialpha 1·GTPgamma S complex because it would be sterically excluded by the gamma -thiophosphate sulfur atom of GTPgamma S. The conformation of Glu43 is altered, and its carboxylate moiety forms a hydrogen bond to W600 and consequently cannot form the hydrogen bond to Arg242 that is present in Gialpha 1·GTPgamma S (Fig. 2, A and B). In Gialpha 1·GppNHp, Glu43 and Arg178 form a doubly hydrogen-bonded ion pair (Fig. 2A), in contrast to the less intimate ion pair interaction observed in the Gialpha 1·GTPgamma S complex (Fig. 2B). This enhanced salt-bridge may strengthen binding of GTP in the ground state. Remarkably, the same interaction occurs in crystals of Gialpha 1·GDP complexed with G protein beta gamma subunits (28). The conformations of Arg178 in Gialpha 1·GppNHp and Gialpha 1·GTPgamma S differ slightly, but in neither case does Arg178 interact with the GTP substrate analog. In contrast, in the Gtalpha ·GTPgamma S complex, the corresponding residue, Arg174, interacts directly with the oxygen bridging the beta - and gamma -phosphate atoms and the gamma -thiophosphate sulfur atom. The bridging NH group of GppNHp would not be capable of this interaction however.


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 1.   Electron density about the active site of the Gialpha 1·GppNHp complex. The 1.5-Å 2 Fo - Fc electron density map (blue) was calculated using SigmaA-weighted phases derived from the model, contoured at 1.5 sigma . The model is shown as a ball-and-stick representation. Red, oxygen; yellow, carbon; blue, nitrogen; green, phosphorous; silver, magnesium. The figure was generated using the program BOBSCRIPT (51) and rendered with RASTER3D (52), and POVRAY.


View larger version (63K):
[in this window]
[in a new window]
 
Fig. 2.   The active site of Gialpha 1 in the (GTP analog) and (transition state analog·RGS4) bound complexes. Key features are labeled. A, Gialpha 1·GppNHp; B, Gialpha 1·GTPgamma S; C, RGS4·Gialpha 1·GDP·AlF4-·H2O; and D, hypothetical RGS4·Gialpha 1·GppNHp complex. The main chain segments of Gialpha 1 are colored green (P-loop residues 38-48), blue (Switch I residues 178-184), and yellow (Switch II residues 200-208). The main chain segment of RGS4 in panels C and D is shown in red (residues 126-131). The atoms and water molecules are colored as described in Fig. 1, except that the phosphorous atoms are in yellow, magnesium is blue, and the sulfur atom of GTPgamma S is green. In panel D, the region of the hypothetical model where Asn128 of RGS4 and Gln204 of Gialpha 1 collide is highlighted in cyan.

The orientation of Gln204 in the GppNHp complex is particularly interesting. In the Gialpha 1·GTPgamma S complex the side chain of this residue, which assumes the favored gauche + chi 1 conformation, is poorly ordered and directed out of the active site, and does not appear to interact with any other residues (Fig. 2B). Similar conformations for the corresponding catalytic glutamine (Glncat) are observed in the GTPgamma S-Gtalpha , -Gsalpha , and -Gsalpha ·adenylyl cyclase complexes (7, 9, 29). In contrast, Gln204 exhibits strong density for the gauche - chi 1 conformation in the Gialpha 1·GppNHp complex and makes several contacts with other moieties (Figs. 1 and 2A). Notably, while Gln204 does not interact directly with the substrate analog, its side chain amino group forms a hydrogen bond to Wnuc. The orientation of the amide group is defined by the functionality of the three other moieties to which Wnuc is hydrogen bonded: the O1G oxygen of the gamma -phosphate group, which is proposed to act as a catalytic base (30, 31), the main chain carbonyl oxygen of Thr181, and the main chain NH group of Gln204 (Fig. 2A). Gln204 is also anchored by a hydrogen bond between its carboxamide oxygen atom and the hydroxyl moiety of Ser206.

The thiophosphate moiety of GTPgamma S does not prevent Gln204 from adopting the conformation observed in the GppNHp complex; however, it does block entry of W600 to the active site. W600, in turn prevents Gln204 from assuming the conformation that is partly populated in the GTPgamma S complex. In the GppNHp complex, the hydrogen bond between the phosphate O1G oxygen and Wnuc is shorter than the corresponding interaction in the GTPgamma S complex (2.82 versus 3.27 Å). The position of Wnuc closer to the gamma -phosphate permits a more favorable interaction with the side chain of Gln204. The well ordered conformation of Gln204 in the GppNHp complex may be attributed to hydrogen bonds formed with Wnuc and Ser206 but also to the conformational restriction imposed by W600 as well as the cryogenic data measurement conditions. In other G protein-GppNHp or -GppCp complexes, Glncat also interacts with Wnuc (32, 33). However, in these cases Glncat assumes the gauche + chi 1 conformation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most intriguing feature of the Gialpha 1·GppNHp complex is the conformation of the side chain of Gln204 and its implications for the mechanism of both intrinsic and RGS-stimulated GTP hydrolysis. As established by mutagenesis studies (11), Gln204, and the corresponding residues in other G proteins (12, 34, 35), is absolutely required for enzymatic activity. Studies of substrate-assisted catalysis using GTPase-deficient Gsalpha have demonstrated that a hydrogen donor group near the gamma -phosphate can substitute for Glncat (36). However, the function of Glncat during GTP hydrolysis by G proteins has been debated. Hydrolysis of GTP in G proteins is believed to occur by a direct, in-line attack on the gamma -phosphate atom by a nucleophilic water (37-39). Crystal structures of Gialpha 1 and other G proteins have identified, near the gamma -phosphate group, a well ordered water molecule (Wnuc) that is positioned to carry out a nucleophilic attack (7-9, 32, 40, 41). Although Gln204 is near Wnuc in Gialpha 1·GTPgamma S, it does not directly contact it. Further, Wnuc is observed in Gln204 right-arrow Leu Gialpha 1·GTPgamma S, and Gln61 right-arrow Leu Ras·GppNHp (Gln61 is Glncat in Ras) (8, 42), indicating that Glncat is not required for binding of Wnuc in the ground state. It has been pointed out that the basicity of glutamine is low, and it is therefore unlikely that Glncat acts as the general base that deprotonates Wnuc (43). Rather, an oxygen of the presumably dianionic gamma -phosphate is proposed to serve this function in Ras (30, 31). Hence, Glncat may only polarize Wnuc in the ground state.

X-ray crystallographic studies have indicated that Glncat stabilizes the transition state (2), as originally proposed by Prive et al. (43). Structures of Gialpha 1 and Gtalpha complexed with the transition state analog GDP·AlF4-, reveal Glncat positioned within the active site and directly interacting with a fluorine substituent and Wnuc of the GDP·AlF4-·Wnuc complex. Fig. 2C shows these interactions in the RGS4·Gialpha 1·GDP·AlF4- complex (44). It was proposed that the amino group of Glncat stabilizes negative character on the equatorial oxygen of the transition state and its carbamoyl oxygen stabilizes the attacking nucleophilic water. A similar configuration is observed in the Ras-GAP·Ras·GDP·AlF4- and the Rho-GAP·Cdc42Hs·GDP·AlF4- complexes (45-47). In addition, mutations that perturb the transition state conformation of Glncat abolish GTPase activity (42, 48). These observations indicate that Glncat stabilizes the transition state for GTP hydrolysis.

The GppNHp complex provides novel insights into both the mechanism of GTP hydrolysis as well as to the role of both Argcat and Glncat in the ground state E·S complex. GppNHp, but not GTPgamma S, permits a water molecule, W600, to occupy a position in which it could act as the ultimate proton acceptor from Wnuc. Water molecules in similar, but not identical, positions are present in the GppNHp- or GppCp-bound complexes of Ras and Rac1 (32, 40). A proton could be relayed from Wnuc to W600 via O1G of the GTP gamma -phosphate. This substituent does not otherwise participate in hydrogen bonds with the protein and corresponds to the thiol of GTPgamma S. The basicity of W600 may be enhanced by hydrogen bond formation with Glu43, which is well conserved in Galpha proteins with the exception of Gzalpha where it is replaced with an asparagine residue. Glu43 also forms a hydrogen-bonded ion pair with Arg178. In this conformation, Arg178 is restrained from interacting with the gamma -phosphate of GTP. Transfer of a proton from Wnuc to W600 would tend to weaken this ion pair, releasing Arg178 to stabilize the incipient pentacoordinate phosphoryl transition state. W600 also blocks the side chain of Gln204 from interacting with the pentacoordinate phosphate. Thus, until it diffuses from the active site, W600 impedes the reorganization of the catalytic site that is required for transition state stabilization.

Gln204 is anchored in a noncatalytic conformation by hydrogen bonds to both Wnuc and Ser206 (Ser206 is substituted by an Asp in Galpha s,). In the ground state, Gln204 could orient and perhaps activate Wnuc; however, to stabilize the transition state as represented by G protein GDP·AlF4- complexes, Gln204 must sever its hydrogen bond with Ser206 and Wnuc and rotate approx 120° about chi 1 and approx 90° about chi 2 and chi 3 (to gauche+ and gauche-, respectively) such that its carbamoyl group donates a hydrogen bond to the equatorial oxygen of the pentacoordinate gamma -phosphoryl group and accepts a hydrogen bond from Wnuc. Such would incur a substantial penalty in catalytic efficiency and perhaps account, at least in part, for the low catalytic rate of GTP hydrolysis in Galpha and perhaps in other G proteins.

We propose that the ground state Gialpha 1·GTP complex is "auto-inhibited" with Glncat locked into an unproductive conformation. Active site residues in the EF-Tu·GppNHp complex also assumes anti-catalytic positions; in this case Hiscat, the residue corresponding to Glncat, cannot interact with the substrates because of steric interference by other active site residues (41). In Gialpha 1, catalysis could occur only if the bonds that hold Glncat in this position are broken, and the side chain freed to interact with the pentacoordinate transition state. This model predicts that changes that disrupt the ground state conformation of Gln204, while not otherwise compromising the active site, would increase kcat.

RGS proteins, which accelerate the rate of GTP hydrolysis by Gialpha 1 by 50-100-fold, may act in part by destabilizing the ground state conformation of Glncat, as well as stabilizing its productive conformation in the transition state (44). The crystal structure of the RGS4·Gialpha 1·GDP·AlF4- complex demonstrates that RGS4 stabilizes the active site of Gialpha 1 in the conformation corresponding to that of the transition state complex (Fig. 2C) (44). No residues from RGS4 are inserted into the active site except Asn128, which could enhance catalysis by aiding in binding, orienting, and polarizing Wnuc in the pre-transition state complex (44).

However, superposition of Gialpha 1·GppNHp and Gialpha 1·GDP·AlF4- from the RGS4·Gialpha 1·GDP·AlF4- complex reveals that the carbamoyl groups of Gln204 and Asn128 occupy nearly the same positions, although the side chains approach from opposite directions (Fig. 2D). Further, both residues are positioned such that they can bind the nucleophilic water and Ser206. We suggest that Asn128 of RGS4 displaces the side chain of Gln204 from its "anti-catalytic" position in the ground state, freeing it to participate in stabilization of the transition state.

Mutational analysis of RGS proteins supports this hypothesis. Mutation of Asn131 in hRGSr (analogous to Asn128 of RGS4) to either serine or glutamine resulted in a relatively small decrease in the kcat of Gtalpha (49). In addition, hRGSr in which Asn131 was mutated to leucine or alanine also retains substantial stimulatory activity, and the loss of activity that was observed could be attributed to weakened binding of these mutants to Gtalpha . Similar mutagenic studies have been performed with RGS4 (50). Mutants of Asn128 analogous to those of hRGSr Asn131 were modeled in the structure of the "RGS4·Gialpha 1·GppNHp" complex. In all cases these residues were in steric conflict with Gln204. These findings indicate that the bulk and binding of the residue at position 128 is important to the stimulatory activity of RGS4 although it is unlikely that it has a direct catalytic role in stimulation of GTPase activity (49).

The evidence presented is consistent with a self-inhibited or anti-catalytic model of the ground state of Galpha proteins, and a role for RGS proteins in stimulating GTPase activity by releasing Galpha subunits from this ground state while stabilizing the transition state.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code1cip) has been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

To whom correspondence should be addressed: Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: Sprang{at}howie.swmed.edu.

    ABBREVIATIONS

The abbreviations used are: GTPgamma S, guanosine 5'-O-3-thiotriphosphate; RGS, regulator of G protein signaling; GppNHp, guanosine-5'-(beta gamma -imino)triphosphate; GppCHp, guanosine-5'-(beta gamma -methylene)triphosphate; Wnuc, nucleophilic water; Glncat and Argcat, conserved catalytic residues in Galpha subunits.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M., and Satoh, T. (1991) Annu. Rev. Biochem. 60, 349-400[CrossRef][Medline] [Order article via Infotrieve]
  2. Sprang, S. R. (1997) Annu. Rev. Biochem. 66, 639-678[CrossRef][Medline] [Order article via Infotrieve]
  3. Deleted in proof
  4. Deleted in proof
  5. Deleted in proof
  6. Taussig, R., Tang, W.-J., Hepler, J. R., and Gilman, A. G. (1994) J. Biol. Chem. 269, 6093-6100[Abstract/Free Full Text]
  7. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663[CrossRef][Medline] [Order article via Infotrieve]
  8. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412[Medline] [Order article via Infotrieve]
  9. Sunahara, R. K., Tesmer, J. J. G., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1943-1947[Abstract/Free Full Text]
  10. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372, 276-279[CrossRef][Medline] [Order article via Infotrieve]
  11. Freissmuth, M., and Gilman, A. G. (1989) J. Biol. Chem. 264, 21907-21914[Abstract/Free Full Text]
  12. Graziano, M. P., and Gilman, A. G. (1989) J. Biol. Chem. 264, 15475-15482[Abstract/Free Full Text]
  13. Deleted in proof
  14. Linder, M. E., Ewald, D. A., Miller, R. J., and Gilman, A. G. (1990) J. Biol. Chem. 265, 8243-8251[Abstract/Free Full Text]
  15. Dohlman, H. G., and Thorner, J. (1997) J. Biol. Chem. 272, 3871-3874[Free Full Text]
  16. Berman, D. M., and Gilman, A. G. (1998) J. Biol. Chem. 273, 1269-1272[Free Full Text]
  17. Lee, E., Linder, M. E., and Gilman, A. G. (1994) Methods Enzymol. 237, 146-164[Medline] [Order article via Infotrieve]
  18. Coleman, D. E., Lee, E., Mixon, M. B., Linder, M. E., Berghuis, A., Gilman, A. G., and Sprang, S. R. (1994) J. Mol. Biol. 238, 630-634[CrossRef][Medline] [Order article via Infotrieve]
  19. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960[Abstract]
  20. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
  21. Read, R. J. (1986) Acta Crystallogr. Sec. A 42, 140-149[CrossRef]
  22. Hodel, A., Kim, S-H., and Brünger, A. T. (1992) Acta Cryst. Sec. A 48, 851-858[CrossRef]
  23. Jones, T. A., and Kjeldgaard, M. O. (1993) O, Version 5.9, Uppsala University, Uppsala, Sweden
  24. Brünger, A. T. (1992) XPLOR, Version 3.1, Yale University Press, New Haven, CT
  25. Jiang, J. S., and Brünger, A. T. (1994) J. Mol. Biol. 243, 100-115[CrossRef][Medline] [Order article via Infotrieve]
  26. Brünger, A. T. (1992) Nature 355, 472-475[CrossRef]
  27. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
  28. Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 80, 1047-1058
  29. Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916[Abstract/Free Full Text]
  30. Schweins, T., Geyer, M., Scheffzek, K., Warshel, A., Kalbitzer, H. R., and Wittinghofer, A. (1995) Nat. Struct. Biol. 2, 36-44[Medline] [Order article via Infotrieve]
  31. Schweins, T., Geyer, M., Kalbitzer, H. R., Wittinghofer, A., and Warshel, A. (1996) Biochemistry 35, 14225-14231[CrossRef][Medline] [Order article via Infotrieve]
  32. Hirshberg, M., Stockley, R. W., Dodson, G., and Webb, M. R. (1997) Nat. Struct. Biol. 4, 147-152[Medline] [Order article via Infotrieve]
  33. Milburn, M. V., Tong, L., deVos, A. M., Brünger, A., Yamaizumi, Z., Nishimura, S., and Kim, S-H. (1990) Science 247, 939-945[Medline] [Order article via Infotrieve]
  34. Der, C. J., Finkel, T., and Cooper, G. M. (1986) Cell 44, 167-176[Medline] [Order article via Infotrieve]
  35. Landis, C. A., Masters, S. B., Spada, A., Pace, A. M., Bourne, H. R., and Vallar, L. (1989) Nature 340, 692-696[CrossRef][Medline] [Order article via Infotrieve]
  36. Zor, T., Andorn, R., Sofer, I., Chorev, M., and Selinger, Z. (1998) FEBS Lett. 433, 326-330[CrossRef][Medline] [Order article via Infotrieve]
  37. Webb, M. R., and Eccleston, J. F. (1981) J. Biol. Chem. 256, 7734-7737[Abstract/Free Full Text]
  38. Eccleston, J. F., and Webb, M. R. (1982) J. Biol. Chem. 257, 5046-5049[Abstract/Free Full Text]
  39. Feuerstein, J., Goody, R. S., and Webb, M. R. (1989) J. Biol. Chem. 264, 6188-6190[Abstract/Free Full Text]
  40. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W., and Wittinghofer, A. (1990) EMBO J. 9, 2351-2359[Abstract]
  41. Berchtold, H., Reshetnikova, L., Reiser, C. O. A., Schirmer, N. K., Sprinzl, M., and Hilgenfeld, R. (1993) Nature 365, 126-132[CrossRef][Medline] [Order article via Infotrieve]
  42. Krengel, U., Schlichting, I., Scherer, A., Schumann, R., Frech, M., John, J., Kabsch, W., Pai, E. F., and Wittinghofer, A. (1990) Cell 62, 539-548[Medline] [Order article via Infotrieve]
  43. Privé, G. G., Milburn, M. V., Tong, L., deVos, A. M., Yamaizumi, Z., Nishimura, S., and Kim, S.-H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3649-3653[Abstract]
  44. Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261[Medline] [Order article via Infotrieve]
  45. Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., and Wittinghofer, A. (1997) Science 277, 333-338[Abstract/Free Full Text]
  46. Rittinger, K., Walker, P. A., Eccleston, J. F., Smerdon, S. J., and Gamblin, S. J. (1997) Nature 389, 758-762[CrossRef][Medline] [Order article via Infotrieve]
  47. Nassar, N., Hoffman, G. R., Manor, D., Clardy, J. C., and Cerione, R. A. (1998) Nat. Struct. Biol. 5, 1047-1052[CrossRef][Medline] [Order article via Infotrieve]
  48. Raw, A. S., Coleman, D. E., Gilman, A. G., and Sprang, S. R. (1997) Biochemistry 36, 15660-15669[CrossRef][Medline] [Order article via Infotrieve]
  49. Natochin, M., McEntaffer, R. L., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 6731-6735[Abstract/Free Full Text]
  50. Posner, B. A., Mukhopadhyay, S., Tesmer, J. J. G., Gilman, A. G., and Ross, E. M. (1999) Biolchemistry, in press
  51. Esnouf, R. M. (1997) J. Mol. Graphics 15, 132-134[CrossRef]
  52. Merritt, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sec. D 50, 869-873[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.