Nature © Macmillan Publishers Ltd 1998 8 NATURE | VOL 394 | 23 JULY 1998 337 articles The structural basis of the activation of Ras by Sos P. Ann Boriack-Sjodin*, S. Mariana Margarit², Dafna Bar-Sagi² & John Kuriyan*‡ * Laboratories of Molecular Biophysics, and ‡ Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA ² Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA ........................................................................................................................................................................................................................................................ The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A ˚ resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. First, the insertion into Ras of an a-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. Second, side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras–Sos complex adopts a structure that allows nucleotide release and rebinding. The Ras proteins are highly conserved guanine-nucleotide-binding enzymes that couple cell-surface receptors to intracellular signalling pathways controlling cell proliferation and differentiation 1,2 . Ras acts as a molecular switch by cycling between active GTP-bound and inactive GDP-bound states. Whether GDP or GTP is bound to Ras is determined by the action of two classes of regulatory proteins: guanine-nucleotide-exchange factors, and GTPase-activating pro- teins. Exchange factors promote the activation of Ras by catalysing the exchange of GDP for GTP, whereas GTPase-activating proteins control the conversion of Ras to the inactive state by stimulating the hydrolysis of GTP to GDP 2 . Cell-surface receptors that signal through tyrosine kinases acti- vate Ras by stimulating the guanine-nucleotide exchange reac- tion 3–5 . Genetic and biochemical studies have indicated that this reaction is controlled by the Ras guanine-nucleotide exchange factor Son of sevenless (Sos) 6 . Following ligand binding, Sos is brought from the cytoplasm to the activated receptor in a phosphotyrosine-dependent manner through adapter proteins such as Grb2. Grb2 contains SH3 domains that are bound con- stitutively to a carboxy-terminal proline-rich region of Sos, and the Grb2–Sos complex is recruited to activated receptors by inter- actions between the SH2 domain of Grb2 and phosphotyrosine residues on the receptor 7 . Because Ras is localized to the membrane, receptor activation results in an increase in the effective concentra- tion of Sos in the vicinity of Ras, facilitating the exchange of bound guanine nucleotide for free cellular guanine nucleotides. The region of Sos (relative molecular mass of about 150,000; M r ,150K) that is functional for nucleotide exchange on Ras spans 1040 ..|....|.... huSos1 PGVRPSNPRPGT DmSos1 PGIKPRRQNQTN Cdc25 KFLSEFTDDKNG Scd25 KSRDDQTDEGKT Ste6 ............ RasGRF ............ 670 680 690 700 ........|....|........|....|....|....|....|....| huSos1 ....IAIENGDQPLS....AELKRFRKEYIQPVQLRVLNVCRHWVEHH DmSos1 GAGGMGGVGGDKEHKNSHREDWKRYRKEYVQPVQFRVLNVLRHWVDHH Cdc25 WIEKKSNPIKCRVVNIMRTFLTQYWTRNYYEPGIPLIL..NFA..KMV Scd25 WVTKKLIPVKCRVVEIMTTFFKQYWFPGYDEPDLATLNLDYFA..QVA Ste6 FISHEFYRVSKRFLDILLIWFESYLVEELDNSKSIFFLFKIYKVFEVF RasGRF ----------------------------------242 residue in 710 720 730 740 750 ....|....|....|....|....|....|....|....|....|....|.. FYDFERDAYLLQRMEEFIGTVRGKAMKKWVESITKIIQRKKIARDNGPGHNI FYDFEKDPMLLEKLLNFLEHVNGKSMRKWVDSVLKIVQRKNEQEKSNKKIVY VSEKIPGAEDL.....LQKINEKLINENEKEPVDPKQQDSVSAVVQTTKRDN IKENITGSVEL.....LKEVNQKFKLGNIQEATAP.....MKTLDQQICQDH VVPHFASAEEL.....LHSLSHLLHHPSTKRSHKMLEGKELSQELEDLSLHN sertion--------------------------------------------- 760 770 780 790 800 810 820 830 840 ..|....|....|....|....|....|....|....|....|....|....|...+++.....+|....|++.+++.++|.++.|+...|....|.... huSos1 TFQSSPPTVEWHISRPGHIETFDLLTLHPIEIARQLTLLESDLYRAVQPSELVGSVWTKE...DKEINSPNLLKMIRHTTNLTLWFEKCIVETENLEERV DmSos1 AYGHDPPPIEHHLSVPN..DEITLLTLHPLELARQLTLLEFEMYKKNVPSELVGSPWTKK...DKEVKSPNLLKIMKHTTNVTRWIEKSITEAENYEERL Cdc25 KSPIHMSSSSLPSSASFRLKKLKLLDIDPYTYATQLTVLEHDLYLRITMFECLDRAWGTKYC.NMGG.SPNITKFIANANTLTNFVSHTIVKQADVKTRS Scd25 YSGTLYSTTE............SILAVDPVLFATQLTILEHEIYCEITIFDCLQKIWKNKYTKSYGA.SPGLNEFISFANKLTNFISYSVVKEADKSKRA Ste6 SPDPIIYKDEL............VLLLPPREIAKQLCILEFQSFSHISRIQFLTKIWDNL...NRFS.PKEKTSTFYLSNHLVNFVTETIVQEEEPRRRT RasGRF TQEEITENHSMLDELLLGVKTEPFENHSAMEIAEQLTLLDHLVFKSIPYEEFFGQGWMKA...DKNERTPYIMKTTRHFNHISNLIASEILRNEEVSARA 850 860 870 880 |....|....|....|...+|.++.+++.++++.++.. huSos1 AVVSRIIEILQVFQELNNFNGVLEVVSAMNSSPVYRLD DmSos1 AIMQRAIEVMMVMLELNNFNGILSIVAAMGTASVYRLR Cdc25 KLTQYFVTVAQHCKELNNFSSMTAIVSALYSSPIYRLK Scd25 KLLSHFIFIAEYCRKFNNFSSMTAIISALYSSPIYRLE Ste6 NVLAYFIQVCDYLRELNNFASLFSIISALNSSPIHRLR RasGRF STIEKWVAVADICRCLHNYNAVLEITSSINRSAIFRLK 890 900 910 920 930 940 ..|....|....|....+....+.++++.|+...|.....|...++++.+++.+++.++++. HTFEQIPSRQKKILEEAHEL..SEDHYKKYLAKLRSIN.PPCVPFFGIYLTNILKTEEGNPE WTFQGLPERYRKFLEECREL..SDDHLKKYQERLRSIN.PPCVPFFGRYLTNILHLEEGNPD KTWDLVSTESKDLLKNLNNLMDSKRNFVKYRELLRSVTDVACVPFFGVYLSDLTFTFVGNPD KTWQAVIPQTRDLLQSLNKLMDPKKNFINYRNELKSLHSAPCVPFFGVYLSDLTFTDSGNPD KTWANLNSKTLASFELLNNLTEARKNFSNYRDCLENCV.LPCVPFLGVYFTDLTFLKTGNDP KTWLKVSKQTKSLFDKLQKLVSSDGRFKNLRETLRNCD.PPCVPYLGMYLTDLAFLEEGTPN 950 ...|........ huSos1 VLKR........ DmSos1 LLA......... Cdc25 FL.........H Scd25 YLVLEHGLKGVH Ste6 ............ RasGRF YT.......... 960 970 980 990 1000 1010 1020 1030 ....|....|..+.|.+..|....|....|....|....|....|....|.++.|.+..++...|...+|....|....|.....|.. HGKELINFSKRRKVAEITGEIQQYQNQPYCLRVESDIKRFFENLNPMGNSMEKEFTDYLFNKSLEIEPRNPKPLPRFPKKYSY.PLKS N.TELINFSKRRKVAEIIGEIQQYQNQPYCLNEESTIRQFFEQLDPFNGLSDKQMSDYLYNESLRIEPRGCKTVPKFPRKWPHIPLKS NSTNIINFSKRTKIANIVEEIISFKRFHYKLKRLDDIQTVIEASLENVP.....HIEKQYQLSLQVEPRSGNTKGSTHASSASGTKTA DEKKYINFNKRSRLVDILQEIIYFKKTHYDFTKDRTVIECISNSLENIP.....HIEKQYQLSLIIEPKP..RKKVVPNSNSNNKNQE NFQNMINFDKRTKVTRILNEIKKFQSVGYMFNPINEVQELLNEVISRER.....NTNNIYQRSLTVEPRESEDQALQRLLIDSGIF.. ..EGLVNFSKMRMISHIIREIRQFQQTTYKIEPQPKVTQYLVDETFVLL.....DDESLYEASLRIEPKLPT................ β2 β1 1301 949 659 1005 620 630 640 650 660 .|....|..#.|##..#...#|....|....|....|....|....|... TYHMYADPNFVRTFLTTYRSFCKPQELLSLIIERFEIPEPEPTEADR... TYHIYADPTFVRTFLTTYRYFCSPQQLLQLLVERFNIPDPSLVYQDTGTA TSHELVDAAFNVTMLITFRSILTTREFFYALIYRYNLYPPEGLSYDDYNI TDNEKKDLFFNITFLITFRSIFTTTEFLSYLISQYNLDPPEDLCFEEYNE LRTDIDSTFFTTIFLNTYASMISSSDLFSILGAHFRFIC..SLNFGKIS. TDLRFLSIDFLNTFLHSYRVTDAVVVLDKLISIYKKPITAIPARSLELLF 565 570 580 590 600 610 .|....|....|....|....|....|....|....|....|#..#|... huSos1 EEQMRLPSADVYRFAEPDSEENIIFEENMQPKAGIPIIKAGTVIKLIERL DmSos1 KHPLRMPSPEIYKFAVPDSGDNIVLEE..RESAGVPMIKGATLCKLIERL Cdc25 LGSEDYIERKAANIEKNLPWFLTSDYETSLVYDSRGKIRGGTKEALIEHL Scd25 ADSDTKDNDEWRDSQVKLPRYLQREYDSELIWGPIVRIKGGSKHALISYL Ste6 KGLVCYLMKQTSPEPLLIRNLLFSFWSCNGKIEQDGKIKTATLVFIINYL RasGRF NAFEENSKVTVPQMIKSDASLYCDDVDIRFSKTMNSCKVLQIRYASVERL 1 α 2 α 3 α 6 α 5 α 4 α B A α α C α D α F G H α α α αE I J K α α α 1121 787 493 633 Figure 1 Sequence alignment of Ras-binding exchange factors with residue numbers of human (hu) Sos1 indicated. The secondary structure elements (a-helices are shown as rectangles, b-sheets as arrows, coil regions as a solid line, disordered residues as broken lines) of the N-domain are dark blue, and those of the catalytic domain are green. Conserved regions are shaded with pale blue. Residues of Sos at the Ras interface are indicated with +; residues in the N-domain that form the hydrophobic core with the catalytic domain are indicated with #. Sequences have been omitted where the sequence similarity between exchange factors is low. DmSos1, Drosophila melanogaster Sos1.