Solution Structure of the SH3 Domain of Phospholipase C-y (1993)

D. Kohda,’ H. Hatanaka,’ M. Odaka,’ V. Mandiyan,t A. Ullrich,* J. Schlessinger,t and F. Inagaki’ *Department of Molecular Physiology The Tokyo Metropolitan Institute of Medical Science 18-22 Honkomagome 3-chome Bunkyo-ku, Tokyo 113 Japan tDepartment of Pharmacology New York University Medical Center New York, New York 10016 *Max-Planck lnstitut ftir Biochemie 8033 Am Klopfrspitz 18A Martinsried Federal Republic of Germany Summary SH3 (Src homology 3) domains are found in many sig- naling proteins and appear to function as binding mod- ules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-y (PLC-1) was determined by two-dimensional ‘ H NMR analysis. This SH3 domain is composed of eight antiparallel p strands consisting of two successive “Greek key” motifs, which form a barrel-like structure. The conserved aliphatic and aromatic residues form a hydrophobic pocket on the molecular surface, and the conserved carboxylic residues are localized to the periphery. The hydrophobic pocket may serve as a binding site for target proteins. Analysis of the slowly exchanging amide protons by NMR measurements in- dicates that despite containing a high content of p structure, the SH3 domain of PLC-y is flexible. InWoduction A large family of growth factors act by binding to and acti- vating cell surface receptors with intrinsic protein-tyrosine kinase activities (reviewed by Ullrich and Schlessinger, 1990). Following ligand-dependent receptor dimerization and tyrosine autophosphorylation, a diverse group of sig- naling proteins, including phospholipase C-y (PLC-y) and GTPase-activating protein of Ras, bind to specific tyro- sine-phosphorylated regions in activated growth factor re- ceptors (reviewed by Cantley et al., 1991; Koch et al., 1991; Schlessinger and Ullrich, 1992). The interaction be- tween these signaling molecules and tyrosine-autophos- phorylated receptors is mediated by a conserved region of approximately 100 amino acids termed Src homology 2 (SH2) domains. Proteins with SH2 domains frequently contain another conserved region of approximately 50 amino acids, the SH3 domain, while other proteins contain SH3 domains independently. SH3 domains are localized in different regions of their host proteins, including the extreme aminoor carboxyl terminus. Interestingly, several small proteins implicated in control of cell proliferation and oncogenesis such as Crk and GRB2/Sem-5, do not have any catalytic domains and are composed virtually of SH2 and SH3 domains (Mayer et al., 1988; Clark et al., 1992; Lowenstein et al., 1992). The emerging picture is that SH3 domains function as independent structural modules par- ticipating in protein-protein interactions involved in cellu- lar signaling. SH3 domains were also found in cytoskeletal proteins such as spectrin, myosin 1, and actin-binding pro- tein of yeast (reviewed by Koch et al., 1991; Drubin et al., 1990), suggesting that these domains may also play a role in protein-protein interactions in the cytoskeleton. It is now well established that the guanine nucleotide- binding protein ~21”” plays a critical role in the control of cell growth and differentiation (Mulcahyet al., 1985; Smith et al., 1986) and that activation of epidermal growth factor, nerve growth factor, and other receptor tyrosine kinases leads to activation of ~21”“. Moreover, it has been shown that a dominant negative mutant of ~21”~ interferes with signaling by receptor tyrosine kinases (Hagag et al., 1986; Szeberenyi et al., 1990; Thomas et al., 1992; Wood et al., 1992). Recent studies have suggested that SH3 domains of certain signaling molecules are involved in the control of the activity of Ras-like guanine nucleotide-binding pro- teins. Semd protein and its human homolog GRBP have been implicated in a highly conserved mechanism for the control of Ras signaling by receptor tyrosine kinases in Caenorhabditis elegans and mammalian cells (Clarket al., 1992; Lowenstein et al., 1992). Consistent with this model, GRB2 or Sem-5 proteins with point mutations in their SH3 domains were impaired in their ability to transmit a biologi- cal signal. Nevertheless, these mutant molecules were capable of binding to activated epidermal growth factor receptors, indicating that interactions with SH3 domains are essential for coupling to a downstream effector mole- cule(s) required for mitogenesis. We have previously proposed that the binding of GRB2 to tyrosine-autophos- phorylated receptors triggers interaction between the SH3 domains of GRB2 and an effector molecule, such as a nucleotide exchange factor, leading to the conversion of the inactive GDP-bound form to the active GTP-bound form of ~21”” (Lowenstein et al., 1992). Consistent with this view is the recent identification of a protein, termed 3BP1, that binds to the SH3 domain of Abl and contains a conserved sequence with similarity to Bcr and GTPase- activating protein-Rho. It is thought that the 3BPl protein functions as a GTPase for Ras-like proteins that is con- trolled by interactions with the SH3 domain of Abl (Cic- chetti et al., 1992), thus providing a mechanism for linking cytoplasmic tyrosine kinase to guanine nucleotide sig- naling. It is important to identify proteins that bind specifically to various SH3 domains of signaling molecules and solve the structures that define specificity of SH3 toward the downstream effector proteins. In this report, we describe the solution structure of SH3 do- main of human PLC-y determined by two-dimensional ‘ H nuclear magnetic resonance (NMR) and stimulated an-