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Crystal Structure of an Active Form of RAS Protein, a Complex of a GTP

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Crystal Structure of an Active Form of RAS Protein, a Complex of a GTP Proc. Natl. Acad. Sci. USA Vol. 87, pp. 4849-4853, June 1990 Biochemistry Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain (RAS protein/G protein/signal transduction/GTP complex of p21) AXEL T. BRUNGER*, MICHAEL V. MILBURNt, LIANG TONGtt, ABRAHAM M. DEVOSt§, JARMILA JANCARIKt, ZIRO YAMAIZUMI¶, SUSUMU NISHIMURA$, EIKO OHTSUKA II, AND SUNG-HOU KIMt** *Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511; tDepartment of Chemistry and Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720; IBiology Division, National Cancer Center Research Institute, Tokyo, Japan; and '1Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Communicated by Melvin Calvin, March 2, 1990 ABSTRACT Normal RAS proteins play a key role of 2.4 mg/ml was incubated in a solution containing 20 mM molecular switch in the transduction of the growth signal from EDTA, apyrase at 2.7 units/ml, and GDP[,8,y-CH21P at 0.17 extracellular to intracellular space. The state of the switch is mg/ml at 370C for 15 min; then 1 M MgSO4 solution was "on" when GTP is bound and "off" when GDP is bound to the added to 33 mM. The mixture was concentrated, excess protein. The crystal structure of a complex between a nonhy- GDP[3,8y-CH2]P was removed, and a buffer solution contain- drolyzable GTP analog and the catalytic domain of a RAS ing 50 mM Hepes, pH 7.5/1 mM EDTA/1 mM dithiothreitol/ protein has been determined by a rotation-translation search 0.001% 1-octylglucoside was added. The last step was re- method. The orientations and positions of four independent peated twice, and the final solution was stored in 20% molecules have been determined using a single molecule as a (vol/vol) glycerol. probe in the search. The crystal structure reveals that the 'y For crystallization the GDP[,8,y-CH2]P complex solution phosphate ofthe GTP analog induces extensive conformational was dialyzed extensively against a solution containing 50 mM changes on two loop regions of the protein. cacodylate buffer, pH 6.5/1 mM dithiothreitol/1 mM EDTA/ 0.01% 1-octylglucoside and concentrated to 14 mg of protein RAS protooncogenes are essential for the cell's growth and per ml in the same buffer. Crystals were obtained from a survival and code for 21-kDa proteins, p21. There are three solution containing GDP[,3,y-CH2]P complex at 7 mg/ml, 100 different RAS protooncogenes located in three different hu- mM magnesium acetate, 10% (vol/vol) polyethylene glycol man chromosomes, and the protein sequences encoded by 8000, and 75 mM cacodylate buffer, pH 6.5, by vapor-phase these genes are practically identical except for the C-terminal equilibrium methods. The space group of the crystals is 25 residues (1). In analogy to elongation factor EF-Tu (2) and P21 (c unique) with unit cell parameters of a = 41.5 A, b = G proteins (3, 4), GTP-complexed RAS proteins are recog- 80.1 A, c = 130.5 A, and 'y = 117.5°. Density measurement nized by effector protein(s) as "on" state, signaling cell growth of GDP[3,8y-CH2]P complex crystals suggested that there are or differentiation, whereas the GDP complexes are perceived three or four GDP[P,y-CH2]P complex molecules per asym- as the signal "off" state. RAS proteins have an intrinsic metric unit of the crystal. Most crystals were layered with GTPase activity to turn off the signal, and this process is each layer slightly misoriented with respect to its neighbors. enhanced by GTPase activating protein, GAP (5-7). X-ray diffraction data were collected on Kodak DEF5 films We have previously determined the crystal structures ofthe using a synchrotron x-ray source at the Brookhaven National GDP complex of a RAS protooncogene product and of a Laboratory. The x-ray wavelength for the data collection was transforming mutant, both lacking the 18 C-terminal residues 1.22 A, and the crystal-to-film distance was 90 mm. All data (8-10). To find out the mechanism of molecular switching, we were collected at 4°C by using an Enraf-Nonius rotation have determined the crystal structure of a complex between a camera. The films were scanned and digitized with an Op- nonhydrolyzable GTP analog, guanosine-5'-(8,y-methylene)- tronix film scanner (Chelmsford, MA) at a resolution of 100 triphosphate (GDP[,3,y-CH2]P), and the normal HRAS protein ,um. The digitized data were processed by using computer lacking the C-terminal 18 residues by using a new molecular programs originally written by Rossmann (13), and the re- replacement method. The structure is refined at 2.5-A reso- flections were reduced and merged by using the PROTEIN lution with a current R factor of 19.5%. The structure reveals program package (14). The entire data set was collected on that an extensive conformational change is induced by the one crystal. During the data collection we searched for and presence of the y phosphate of GDP[,,y-CH2]P, which estab- irradiated the nonmultiple portion of a crystal. A total of lishes additional hydrogen bonding interactions with two sep- 74,084 observations were made to a resolution of 2.5 A, arate loop regions of the protein. At present resolution and which reduced to give 21,061 unique reflections. Average state of refinement, except residues 62-65, the overall struc- Rsym(I) was 6.1% and Rmerge(I) was 8.8%. tural features are similar to the structure of the complex between RAS protein lacking the C-terminal 23 residues Structure Determination complexed with another nonhydrolyzable GTP analog, gua- nosine-5'-(J3,y-imido)triphosphate (GDP[13,y-NH]P) and re- Conventional rotation-translation search methods (15) did fined to 22.9% by using 10.0- to 2.6-A resolution data (11). not provide a unique solution, although one (possibly two) of Cloning, expression, and purification of the protein have been described earlier (12). The Escherichia coli-expressed Abbreviations: PC, Patterson correlation; GAP, GTPase activating protein contained GDP. To exchange this for the nonhydro- protein; GDP(/3,y-,CH2]P, guanosine-5'-(3,'-<methylene)triphosphate; lyzable GTP analog, GDP[3,'y-CH2]P, the GDP complex at GDP[3,y-NH]P, guanosine-5'-(,8y-imido)triphosphate. tPresent address: Department of Biological Sciences, Purdue Uni- versity, West Lafayette, IN 47907. The publication costs of this article were defrayed in part by page charge §Present address: Department of Biomolecular Chemistry, Genen- payment. This article must therefore be hereby marked "advertisement" tech, Inc., South San Francisco, CA 94080. in accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 4849 Downloaded by guest on September 30, 2021 4850 Biochemistry: Brunger et al. Proc. Natl. Acad. Sci. USA 87 (1990) the four rotation solutions was identified in retrospect. This 4 b is understandable because there are three or four molecules 3 1 I _ a in an asymmetric unit. By applying a more recently devel- X 12 ~~~~~15-4 oped molecular-replacement method (16) the structure was I_ successfully solved.tt A brief description of the steps taken 0 follows. Rotation Search. (i) A self-rotation search was performed A B to locate noncrystallographic symmetry elements using a 0.100 ffi«C D _ b | 4 real-space Patterson search method (17). The Patterson map 40 0.075 ffi ffi Average- was calculated on a 1-A grid by using reflections 0.050 all from 15- 0.025 to 4-A resolution. Patterson vectors were selected according 0.000 II to length (between 24 and 5 A). The selected Patterson 0.100 I ,I vectors were rotated using the spherical polar angles 4, c (4i, 0 0.075 15-4i-4 I_ K), and the product correlation with the Patterson map was Pi 0.050 computed by linear eight-point interpolation. 0.025 ll~APAd-A[KW% The self- Ii ,\ I rotation search was carried out by varying 4i and 4 in steps 0.000 ~~~~.7T ~~~~~~:VV '-i-~vX* -,I-1 = 0° = 0.100 L 1 l_ of 20 in the angular range 4i to 180° and 4 00 to 1800, _ A: d u 0.075 ~~~~~~~~~~15-31 - while K was held fixed at 1800. Apart from the crystallo- 04 0.050 I I -7 graphic diad at 4, = 900, a strong noncrystallographic 2-fold 0.025 axis emerges at 4 = 600, ) = 00, and three other positions (4i 0.000 = 1500, 4 = 00; 4 = 300, 4 = 1800; 4 = 1200, 4 = 1800) that 0.100 c e are related to each other by crystallographic symmetry. This 0.075 15-2.5 . - strong, unique peak suggests that there is an even number of 0 0.050 0.025 molecules, four rather than three, in the asymmetric unit, and 0.000 I I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I that the four molecules form two dimers where the monomers 1 25 50 75 100 125 in each dimer (or the two dimers) are related by the non- crystallographic 2-fold axis. A search for a noncrystallo- RF Peak Index graphic 4-fold axis did not produce a unique symmetry element. FIG. 1. By using the crystal structure of the GDP complex of normal human HRAS p21 catalytic domain minus GDP and residues (ii) We performed a rotation search using the crystal 30-40 and 60-70 as probe, a rotation search was performed on structure of GDP complex of the catalytic domain of normal diffraction data of the GDP[L,y-CH2]P complex of the protein.
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