Src homology adaptor proteins: more than the sum of the parts?

Abstract

The recent determination of the crystal structure of the 25 kDa growth factor receptor-bound protein 2 (Grb2) [1xCrystal structure of the mammalian Grb2 adaptor. Maignan, S., Guilloteau, J.-P., Fromage, N., Arnoux, B., Becquart, J., and Ducruix, A. Science. 1995; 268: 291–293Crossref | PubMedSee all References[1] brings us a step further on the road of discovery outlined by Francois Jacob. He described biological macromolecules as having evolved by “combining several systems to produce a more complex one”, a process which “resembles not engineering but tinkering”, and in which “Nature operates to create diversity by endlessly combining bits and pieces” [2xJacob, F. See all References[2]. Grb2 is an example of an apparently ‘tinkered’ molecule. A member of the class of Src homology (SH) domain-containing proteins known as ‘adaptors’, Grb2 has three SH domains in the order SH3–SH2–SH3.Adaptor proteins have no apparent catalytic transcriptional activity, in contrast to other classes of proteins containing SH2 and SH3 domains. The first member of the adaptors identified was the oncogene product, Crk [3xA novel viral oncogene with structural similarity to phospholipase C. Mayer, B.J., Hamaguchi, M., and Hanafusa, H. Nature. 1988; 332: 272–275Crossref | PubMedSee all References[3], and Grb2 is one of the best biologically understood adaptors, playing a key role in the pathway linking activation of cell-surface receptors to Ras activation, and subsequent modification of mitogen-activated protein (MAP) kinases and transcriptional regulation (reviewed in [[4]xSH2 and SH3 domains. Pawson, T. and Schlessinger, J. Curr. Biol. 1993; 3: 434–442Abstract | Full Text PDF | PubMed | Scopus (460)See all References, [5]xThe GRB2/Sem-5 adaptor protein. Downward, J. FEBS Lett. 1994; 338: 113–117Abstract | Full Text PDF | PubMed | Scopus (143)See all References]). Grb2 binds to a range of tyrosine-phosphorylated targets, such as the epidermal growth factor receptor, via its SH2 domain, and to its downstream target, the guanine nucleotide exchange factor, Son of sevenless (Sos), via its SH3 domains. The structures of isolated SH3 domains from human Grb2 and homologs from other species, including their ligated forms [[6]xOrientation of the fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy. Wittekind, M...., and Mueller, L. Biochemistry. 1994; 33: 13531–13539Crossref | PubMed | Scopus (74)See all References, [7]xNMR structure of the N-terminal SH3 domain of Grb2 and its complex with a proline-rich peptide from Sos. Goudreau, N...., and Roques, B.P. Nat. Struct. Biol. 1994; 1: 898–907Crossref | PubMed | Scopus (89)See all References, [8]xCritical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition. Lim, W.A. and Richards, F.M. Nat. Struct. Biol. 1994; 1: 221–225Crossref | PubMed | Scopus (74)See all References, [9]xStructure of the N-terminal SH3 domain of Grb2 complexed with a peptide from the guanine nucleotide release factor, Sos. Terasawa, H...., and Inagaki, F. Nat. Struct. Biol. 1994; 1: 891–897Crossref | PubMed | Scopus (86)See all References, [10]xSolution structure and ligand-binding site of the carboxy-terminal SH3 domain of Grb2. Kohda, D...., and Inagaki, F. Structure. 1994; 2: 1029–1040Abstract | Full Text | Full Text PDF | PubMed | Scopus (63)See all References, [11]xThe crystal structure of the N-terminal SH3 domain of Grb2. Guruprasad, L., Dhanaraj, V., Timm, D., Blundell, T.L., Gout, I., and Waterfield, M.D. Nat. Struct. Biol. 1995; 2: in pressSee all References], have been reported. Apparently, no allosteric communication occurs between SH2 and SH3 domains on binding to their respective ligands [12xIndependent binding of peptide ligands to the SH2 and SH3 domains of Grb2. Lemmon, M.A., Ladbury, J.E., Mandiyan, V., Zhou, M., and Schlessinger, J. J. Biol. Chem. 1994; 269: 31653–31658PubMedSee all References[12] in the complete Grb2 molecule. These, and other, data [13xMembrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Aronheim, A., Engelberg, D., Li, N., al-Alawi, N., Schlessinger, J., and Karin, M. Cell. 1994; 78: 949–961Abstract | Full Text PDF | PubMed | Scopus (372)See all References[13] suggest that the principal action of Grb2 is the recruitment of the Grb2–Sos complex to the plasma membrane in the vicinity of the activated receptor, followed by formation of a multicomponent complex with membrane-bound Ras.The crystal structure of Grb2 [1xCrystal structure of the mammalian Grb2 adaptor. Maignan, S., Guilloteau, J.-P., Fromage, N., Arnoux, B., Becquart, J., and Ducruix, A. Science. 1995; 268: 291–293Crossref | PubMedSee all References[1] generally supports these ideas, and also raises a few intriguing issues. The protein is shown to comprise three distinct domains, with considerable separation of the SH3 domains from the SH2 domain by an interlaced junction (Figure 1Figure 1). There appears to be a better-defined interface between the two SH3 domains (Figure 2Figure 2), although owing to the relatively small area of buried surface (1000 a2), the authors suggest that this may permit dissociation of the two SH3 domains and adoption of different conformations, with positioning of the SH3 segments restricted solely by the length and flexibility of the linker segments (residues 56–59 and 152–158) as illustrated schematically in Figure 3Figure 3.Figure 1Ribbon diagram of Grb2. The three SH domains are labeled, with SH3-C and SH3-N denoting the C- and N-terminal SH3 domains, respectively. (Reproduced from [1xCrystal structure of the mammalian Grb2 adaptor. Maignan, S., Guilloteau, J.-P., Fromage, N., Arnoux, B., Becquart, J., and Ducruix, A. Science. 1995; 268: 291–293Crossref | PubMedSee all References[1]).View Large Image | View Hi-Res Image | Download PowerPoint SlideFigure 2Interface between the two SH3 domains of Grb2. Atoms in contact are represented by space-filling model. (Figure courtesy of Sebastien Maignan.)View Large Image | View Hi-Res Image | Download PowerPoint SlideFigure 3Schematic representation of the possible relative orientations of the N-terminal (N) and C-terminal (C) SH3 domains of Grb2. Arrows indicate the distance between SH3 peptide binding sites. (a) Closed: as observed in Grb2 X-ray structure; (b) half open; (c) open. (Adapted from [1xCrystal structure of the mammalian Grb2 adaptor. Maignan, S., Guilloteau, J.-P., Fromage, N., Arnoux, B., Becquart, J., and Ducruix, A. Science. 1995; 268: 291–293Crossref | PubMedSee all References[1].)View Large Image | View Hi-Res Image | Download PowerPoint SlideThe relative orientations of the SH2 and SH3 domains appear quite different from that of the regulatory apparatus [SH(3+2)] of Lck, reported previously [14xStructure of the regulatory domains of the Src-family tyrosine kinase Lck. Eck, M.J., Atwell, S.K., Shoelson, S.E., and Harrison, S.C. Nature. 1994; 368: 764–769Crossref | PubMed | Scopus (174)See all References[14]. There has been considerable speculation that the positioning of SH2 and SH3 domains may have special structural significance, mainly on the basis of the frequent juxtapositioning of the domain types in different proteins, and the relatively high sequential conservation of the linker region among families of related SH3- and SH2-containing proteins. Whereas the Grb2 results do not exclude the possibility that the linkers have special functions in some families, it seems unlikely that there is any general purpose to these linker regions, other than to bring together the various ligand-containing partners in the intracellular signaling complex.The crystal structure of Grb2 contains two molecules in the asymmetric unit with a local two-fold axis. A substantial dimer interface (4100 a2) and the lack of overlap of this interface with putative binding sites for ligands to the SH2 and SH3 domains raises the question that this dimerization has some functional role. This is reminiscent of the suggested role for dimerization in the Lck kinase regulation [14xStructure of the regulatory domains of the Src-family tyrosine kinase Lck. Eck, M.J., Atwell, S.K., Shoelson, S.E., and Harrison, S.C. Nature. 1994; 368: 764–769Crossref | PubMed | Scopus (174)See all References[14]. Both Grb2 and Lck SH(3+2) are observed as dimers in the crystal form, but not, apparently, in solution. As both proteins are membrane associated, could this relatively weak dimerization have a significant role in biological activity, by, for example, limiting the dissociation of complexes incompletely occupied by their full set of ligands?While such speculations are intriguing, we should probably recall Jacob's concept of molecular evolution [2xJacob, F. See all References[2], and put our bets on a methodical test of these and other notions. The paper by Maignan et al., by providing the first description of a multiple SH domain-containing protein in its entirety, not only represents a significant advance in our knowledge of Grb2, but may provide insights into complex adaptor proteins in general and related multiple SH and other domain-containing proteins.