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• W2145655630 abstract "Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cε, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase γ (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site ofSaccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674–1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclasein vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2Val-19)-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals. Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cε, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase γ (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site ofSaccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674–1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclasein vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2Val-19)-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals. leucine-rich repeat adenylyl cyclase-associated protein Ras-associating domain Ral guanine nucleotide dissociation stimulator phospholipase C Ras-binding domain phosphoinositide 3-kinase glutathione S-transferase guanosine 5′-O-(3-thiotriphosphate) guanosine 5′-O-(2-thiodiphosphate) 2-(N-morpholino)ethanesulfonic acid Ras proteins are small guanine nucleotide-binding proteins cycling between the active GTP-bound and the inactive GDP-bound states. They are conserved from yeasts to mammals and essential signaling components regulating a number of biological responses. The budding yeastSaccharomyces cerevisiae has two RAS genes,RAS1 and RAS2, that encode proteins highly homologous to mammalian Ras proto-oncogene products (1Gibbs J.B. Marshall M. Microbiol. Rev. 1989; 53: 171-185Crossref PubMed Google Scholar, 2Broach J.R. Deschenes R.J. Adv. Cancer Res. 1990; 54: 79-139Crossref PubMed Scopus (161) Google Scholar). The yeast RAS proteins are essential regulatory elements of adenylyl cyclase, which catalyzes the production of cAMP, a second messenger vital for yeast cell growth. The Ras-cAMP pathway is implicated in transduction of glucose-triggered signals to intracellular environments where cAMP initiates protein phosphorylation cascades. Yeast cells bearing the activated RAS2 gene, RAS2Val-19, exhibit an elevated level of intracellular cAMP and display abnormal phenotypes, including sensitivity to heat shock, sensitivity to nutritional starvation, and failure to sporulate (3Kataoka T. Powers S. Cameron S. Fasano O. Goldfarb M. Broach J. Wigler M. Cell. 1985; 40: 19-26Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 4Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (706) Google Scholar). Mammalian Ras proteins can substitute for yeast RAS to induce activation of S. cerevisae adenylyl cyclase (3Kataoka T. Powers S. Cameron S. Fasano O. Goldfarb M. Broach J. Wigler M. Cell. 1985; 40: 19-26Abstract Full Text PDF PubMed Scopus (169) Google Scholar, 5DeFeo-Jones D. Tatchell K. Robinson L.C. Sigal I.S. Vass W.C. Lowy D.R. Scolnick E.M. Science. 1985; 228: 179-184Crossref PubMed Scopus (103) Google Scholar, 6Broek D. Samiy N. Fasano O. Fujiyama A. Tamanoi F. Northup J. Wigler M. Cell. 1985; 41: 763-769Abstract Full Text PDF PubMed Scopus (195) Google Scholar).S. cerevisiae adenylyl cyclase consists of 2,026 amino acid residues and includes at least four domains: the N-terminal, middle repetitive, catalytic, and C-terminal domains (7Kataoka T. Broek D. Wigler M. Cell. 1985; 43: 493-505Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 8Yamawaki-Kataoka Y. Tamaoki T. Choe H.-R. Tanaka H. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5693-5697Crossref PubMed Scopus (82) Google Scholar). The middle repetitive domain is composed of a repetition of a 23-amino acid amphipathic leucine-rich motif and hence is called the LRR1 domain. Introduction of insertion or point mutations into virtually any LRR unit abrogated RAS2-dependent activation of adenylyl cyclase, implicating the LRR domain as a primary Ras interaction site (9Colicelli J. Field J. Ballester R. Chester N. Young D. Wigler M. Mol. Cell. Biol. 1990; 10: 2539-2543Crossref PubMed Scopus (36) Google Scholar, 10Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar, 11Wang J. Suzuki N. Kataoka T. Mol. Cell. Biol. 1992; 12: 4937-4945Crossref PubMed Scopus (19) Google Scholar). Adenylyl cyclase forms a complex with 60-kDa CAP through its C-terminal region (12Field J. Vojtek A. Ballester R. Bolger G. Colicelli J. Ferguson K. Gerst J. Kataoka T. Michaeli T. Powers S. Riggs M. Rodgers L. Wieland I. Wheland B. Wigler M. Cell. 1990; 61: 319-327Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 13Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar, 14Nishida Y. Shima F. Sen H. Tanaka Y. Yanagihara C. Yamawaki-Kataoka Y. Kariya K. Kataoka T. J. Biol. Chem. 1998; 273: 28019-28024Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). CAP is a multifunctional protein. Its N-terminal region is required for the proper function of the Ras-cAMP pathway, while its C-terminal region is involved in regulation of the actin cytoskeleton (13Wang J. Suzuki N. Nishida Y. Kataoka T. Mol. Cell. Biol. 1993; 13: 4087-4097Crossref PubMed Scopus (26) Google Scholar, 15Gerst J.E. Ferguson K. Vojtek A. Wigler M. Field J. Mol. Cell. Biol. 1991; 11: 1248-1257Crossref PubMed Scopus (116) Google Scholar, 16Freeman N.L. Chen Z. Horenstein J. Weber A. Field J. J. Biol. Chem. 1995; 270: 5680-5685Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).We previously demonstrated that at least two kinds of interaction with Ras are required for efficient activation of adenylyl cyclase (17Shima F. Yamawaki-Kataoka Y. Yanagihara C. Tamada M. Okada T. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (47) Google Scholar, 18Shima F. Okada T. Kido M. Sen H. Tanaka Y. Tamada M. Hu C.-D. Yamawaki-Kataoka Y. Kariya K. Kataoka T. Mol. Cell. Biol. 2000; 20: 26-33Crossref PubMed Scopus (59) Google Scholar). One is a GTP-dependent high affinity interaction between Ras and the LRR domain. The other is a GTP-independent weak interaction between Ras and a complex of adenylyl cyclase and CAP, which is dependent upon post-translational modification, in particular farnesylation, of Ras. The latter interaction successfully explains the reason why farnesylation of Ras is essential for the efficient activation of adenylyl cyclase by Ras (19Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (119) Google Scholar).Recently a computer algorithm-based study by Ponting and Benjamin (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (177) Google Scholar) revealed the existence of a motif of roughly 100 amino acid residues, called RAD, which was conserved among Ras-binding regions of mammalian RalGDS, Rin1, and afadin/AF-6 (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (177) Google Scholar). A Ras homologue Rap1, which possesses the identical effector region with Ras, was shown to bind to almost all of these RADs as well. Subsequent studies identified RADs in a variety of other Ras/Rap1 effector candidates including PLCε (21−24) and RA-GEF-1 (25Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 26Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Interestingly, x-ray crystallographic studies revealed that the overall tertiary structure of RalGDS RAD is similar to those of Raf-1 and PI3-Kγ RBDs, although no extensive sequence similarity is found among them (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 28Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar, 29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 32Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 1999; 402: 313-320Crossref PubMed Scopus (410) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar). These studies as well as various mutational studies on Ras revealed that the effector regions of Ras and Rap1 (amino acid 32–40 of mammalian Ras/Rap1) are mainly responsible for the associations with the RAD/RBDs. Although the studies described above implied a universal nature of RADs as primary Ras-binding sites of the Ras effectors, yeast adenylyl cyclase comprised a notable exception because its LRR domain did not appear to bear any resemblance to RADs.In this study, a computer algorithm-based search has unexpectedly predicted a RAD in the N-terminal part of the LRR domain of yeast adenylyl cyclase. Evidence is presented for the essential function of the RAD in association with Ras.DISCUSSIONAlthough genetic and biochemical studies by this and other groups (9Colicelli J. Field J. Ballester R. Chester N. Young D. Wigler M. Mol. Cell. Biol. 1990; 10: 2539-2543Crossref PubMed Scopus (36) Google Scholar, 10Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar) previously indicated the crucial role of the LRR structure in Ras-dependent activation of adenylyl cyclase, the present study has clearly demonstrated that the RAD located in amino acids 676–756, overlapping with the LRR structure, is responsible for the direct association with Ras. This was proven by observation of the GTP-dependent association of human Ha-Ras with the RAD polypeptide with an affinity comparable to that of the whole LRR domain polypeptide. The binding activity of the RAD to yeast RAS2 was demonstrated by its ability to suppress the RAS2Val-19-dependent heatshock sensitivity when overexpressed in yeast cells. Further, specific mutations of the RAD, which abolished its Ras binding activity, were shown also to abrogate the in vitro response of adenylyl cyclase to Ras protein. These results are in good agreement with our previous data, which indicated that N-terminal deletions down to amino acid 657 had no effect on the response of adenylyl cyclase to Ras (10Suzuki N. Choe H.-R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar). The detrimental effect of various mutations affecting the LRR structure on Ras-dependent activation, which we previously observed, appears indirect: it may be ascribed to a change in conformations of adenylyl cyclase, which may affect the allosteric transmission of the Ras-binding signal to the C-terminal catalytic domain. In fact, adenylyl cyclase molecules carrying these mutations were observed to form a high molecular weight complex, whose size was considerably greater than that of the wild-type adenylyl cyclase, which may reflect a change in the oligomeric state of adenylyl cyclase (11Wang J. Suzuki N. Kataoka T. Mol. Cell. Biol. 1992; 12: 4937-4945Crossref PubMed Scopus (19) Google Scholar).RADs were discovered through a computer algorithm-based analysis by Ponting and Benjamin (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (177) Google Scholar) as domains of about 100 amino acids whose primary structures were conserved in a variety of Ras/Rap1 effectors and effector candidates including mammalian RalGDS and its homologues Rlf and Rgl, afadin/AF-6, and its Drosophila melanogaster homologue Canoe and Rin1 (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (177) Google Scholar). Subsequent studies identified RADs in Ras/Rap1 effector candidates including PLCε and RA-GEF-1 (21Song C. Hu C.-D. Masago M. Kariyai K. Yamawaki-Kataoka Y. Shibatohge M. Wu D. Satoh T. Kataoka T. J. Biol. Chem. 2001; 276: 2752-2757Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 22Lopez I. Mak E.C. Ding J. Hamm H.E. Lomasney J.W. J. Biol. Chem. 2001; 276: 2758-2765Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 23Kelley G.G. Reks S.E. Ondrako J.M. Smrcka A.V. EMBO J. 2001; 20: 743-754Crossref PubMed Scopus (298) Google Scholar, 24Jin T.-G. Satoh T. Liao Y. Song C. Gao X. Kariya K. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 30301-30307Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 25Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 26Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The RADs of these effector and effector candidates exactly corresponded to their GTP-dependent Ras/Rap1-binding sites. The RAD sequences were extremely divergent with one another and required sensitive profile methods for detection. This extreme sequence divergence suggested possible divergence of their binding partners. In fact, the RADs of the Ras/Rap1 effectors exhibited mutually distinct binding specificities toward various effector region mutants of Ras as well as toward Ras and Rap1. For example, RalGDS RAD binds more strongly to Rap1 than to Ras, whereas Raf-1 RBD exhibits an opposite binding specificity toward Rap1 and Ras (40Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (298) Google Scholar, 41Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. FEBS Lett. 1999; 451: 175-180Crossref PubMed Scopus (82) Google Scholar). Moreover, some RADs that bound small GTPases other than Ras and Rap1 were identified: the RADs of RA-GEF-2 and GFR/MR-GEF were reported to bind specifically to M-Ras (R-Ras3) but not to Ras and Rap1 (42Ichiba T. Hoshi Y. Eto Y. Tajima N. Kuraishi Y. FEBS Lett. 1999; 457: 85-89Crossref PubMed Scopus (26) Google Scholar, 43Rebhun J.F. Castro A.F. Quilliam L.A. J. Biol. Chem. 2000; 275: 34901-34908Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 44Gao X. Satoh T. Liao Y. Song C. Hu C.-D. Kariya K. Kataoka T. J. Biol. Chem. 2001; 276: 42219-42225Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The RAD of AF-6/Afadin was reported to bind to M-Ras as well as to Ras and Rap1(45). On the other hand, the amino acid sequences of the RBDs of Raf-1 and PI3-Kγ did not score high with the RAD searching program. However, it was found that the majority of hydrophobic residues of Raf-1 RBD were conserved with the RAD sequences and that the predicted secondary structure of Raf-1 RBD using the fold-recognition algorithm (46Russell R.B. Copley R.R. Barton G.J. J. Mol. Biol. 1996; 259: 349-365Crossref PubMed Scopus (115) Google Scholar) matched those of RADs (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (177) Google Scholar). In fact, it was demonstrated by a series of x-ray crystallographic studies that the tertiary structures of RBDs of Raf-1 and PI3-Kγ display a considerable similarity to that of RalGDS RAD despite the quite limited identities in their primary structures (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 28Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar, 29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 32Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 1999; 402: 313-320Crossref PubMed Scopus (410) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar).The amino acid sequences and the known or predicted secondary structures of the RBDs of Raf-1 and PI3-Kγ and the RADs of other Ras/Rap1 effectors are shown in Fig. 6. The crystal structures of Raf-1 RBD complexed with Rap1 and RalGDS RAD complexed with Ras revealed that the first two β-strands (β1 and β2), the next α-helix (α1), and a following loop are important for the interaction with the small GTPases (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 41Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. FEBS Lett. 1999; 451: 175-180Crossref PubMed Scopus (82) Google Scholar). In general, the interactions are mediated mainly by hydrogen bonds and polar interactions formed by charged residues and both main-chain and side-chain polar groups. Only a few hydrophobic contacts are formed. In case of RalGDS, the major interaction between Ras and its RAD occurs between two antiparallel β-strands; β2 of Ras including the switch I/effector region and β2 of the RAD, using hydrogen-bond networks formed between these two β-strands (29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar). Raf-1 RBD also employs a quite similar mechanism for interaction with Rap1 (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 28Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar). In particular, complementarity of the electrostatic charge distribution at the interfaces is shown to be important. The effector region of Ras/Rap1 is acidic and generates a negatively charged surface patch that interacts with a positively charged patch generated by basic residues of the RAD/RBDs (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 28Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar, 29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 32Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 1999; 402: 313-320Crossref PubMed Scopus (410) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar, 41Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. FEBS Lett. 1999; 451: 175-180Crossref PubMed Scopus (82) Google Scholar). Indeed, previous mutational analyses indicated that some of these charged residues were crucial for binding to Ras/Rap1 (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 28Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar, 29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 32Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 1999; 402: 313-320Crossref PubMed Scopus (410) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar).However, there exist notable differences in the mechanisms by which Ras and Rap1 recognize RalGDS, Raf-1, and PI3-Kγ. We shall discuss the differences of the recognition mechanisms in terms of the distribution of the critical basic residues in the β1-, β2-strands, α1-helix, and a loop of the RAD/RBDs and of the interaction mechanism to the various effector region residues of Ras/Rap1. In case of rat RalGDS RAD, three basic residues; Arg-784, Lys-796, and Lys-816 (shown bybold face letters in Fig. 6), form a major binding interface with Ras/Rap1. Arg-784 makes an ionic interaction with Glu-37 of Ras/Rap1 (31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar, 41Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. FEBS Lett. 1999; 451: 175-180Crossref PubMed Scopus (82) Google Scholar). Lys-796 and Lys-816 interact with Glu-38 of Ras/Rap1: Lys-796 makes an ionic interaction with Asp-38, while Lys-816 makes a tight hydrogen bond with Asp-38 to form a main interface with Asp-38 (29Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 30Geyer M. Herrmann C. Wohlgemuth S. Wittinghofer A. Kalbitzer H.R. Nat. Struct. Biol. 1997; 4: 694-699Crossref PubMed Scopus (100) Google Scholar, 31Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (204) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar, 41Vetter I.R. Linnemann T. Wohlgemuth S. Geyer M. Kalbitzer H.R. Herrmann C. Wittinghofer A. FEBS Lett. 1999; 451: 175-180Crossref PubMed Scopus (82) Google Scholar). When the primary structures of RalGDS RAD and two RADs (N-terminal and C-terminal RADs) of afadin/AF-6 were aligned with one another, all of the three basic residues corresponding to Arg-784, Lys-796, and Lys-816 of RalGDS RAD were conserved in both N-terminal and C-terminal RADs (Fig.6A), suggesting that afadin/AF-6 RADs may be classified into the RalGDS RAD group.On the other hand, Raf-1 RBD exhibits a different mode of interaction with Ras/Rap1 for the following reasons. Arg-59 corresponds to Arg-784 of RalGDS in the β1-strand, but Arg-59 makes a strong hydrogen bond with Glu-37 of Ras/Rap1 in contrast to the ionic interaction of Arg-784. Strikingly, Raf-1 RBD lacks two Lys residues corresponding to Lys-796 and Lys-816 of RalGDS in its β2-strand and α1-helix, respectively. Instead, Arg-89 forms a hydrogen bond with Asp-38 of Ras/Rap1 and makes a main binding surface in the α1-helix, thereby functionally substituting for Lys-816 of RalGDS (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar). Although two Lys residues Lys-84 and Lys-87 exist in the α1-helix of Raf-1 RBD, they do not contribute to the interaction with Asp-38 of Ras (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar). A notable difference exists at the binding surface of the β2-strand. There exists no basic residue corresponding to Lys-796 of RalGDS, and its function is replaced by Thr-68, whose side-chain hydroxyl group forms a hydrogen bond with Asp-38 of Ras/Rap1 (27Nassar N. Horn G. Herrmann H. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (558) Google Scholar, 33Pacold M.E. Suire S. Perisic O. Lara-Gonzalez S. Davis C.T. Walker E.H. Hawkins P.T. Stephens L. Eccleston J.F. Williams R.L. Cell. 2000; 103: 931-943Abstract Full Text Full Text PDF PubMed Google Scholar).The crystal structure of PI3-Kγ RBD complexed with Ras revealed that PI3-Kγ RBD also exhibits a distinct mode of interaction with Ras/Rap1 from RalGDS and Raf-1 (32Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 199" @default.
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