COMMENTARY Signal Transduction and Cellular Organization 1253 SH3 domains: complexity in moderation Bruce J. Mayer Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030- 3301, USA ([email protected]) Journal of Cell Science 114, 1253-1263 © The Company of Biologists Ltd Summary The SH3 domain is perhaps the best-characterized member adaptable means of bringing proteins together. The wealth of the growing family of protein-interaction modules. By of genetic, biochemical and structural information binding with moderate affinity and selectivity to proline- available provides an intimate and detailed portrait of the rich ligands, these domains play critical roles in a wide domain, serving as a framework for understanding other variety of biological processes ranging from regulation of modular protein-interaction domains. Processes regulated enzymes by intramolecular interactions, increasing the by SH3 domains also raise important questions about the local concentration or altering the subcellular localization nature of specificity and the overall logic governing of components of signaling pathways, and mediating the networks of protein interactions. assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests Key words: Protein-protein interaction, Modular binding domains, that they provide the cell with an especially handy and Signal transduction Introduction tyrosine kinases, the Crk adaptor protein, and phospholipase The idea that specific protein-protein interactions govern many C-γ (Mayer et al., 1988; Stahl et al., 1988). It was soon biological processes is by now so deeply ingrained that it is apparent that this ~60-residue region of similarity is present in easy to forget that it has only been a decade or so since this many proteins (at this point data from genomic sequencing was widely appreciated. In that time the Src homology 3 (SH3) projects suggest that 63 proteins in Drosophila, 55 in domain has become a standard-bearer for the protein Caenorhabditis elegans, and 25 in Saccharomyces cerevisiae interaction field, largely because it was one of the first modular contain at least one copy of the domain (Rubin et al., 2000); protein interaction domains identified, is one of the most for a graphical representation of the domain structure of the common and has served as a proving ground for new methods ~1000 proteins in the database containing SH3 domains, aimed at identifying partners or creating novel ligands through browse the Pfam database [http://pfam.wustl.edu/] (Bateman et chemical synthesis. Indeed, given the unrelenting attention to al., 1999)). Because the small size of the module seemed to such a modest domain, it seems hard to imagine that there preclude enzymatic activity, the search for function naturally could be much left to uncover. I will argue that, to the contrary, focused on potential protein interactions, and screening of SH3 domains actually serve to remind us just how much expression libraries using isolated SH3 domains soon remains to be learned. In fact their very omnipresence is one identified seemingly specific binding partners (Cicchetti et al., source of conceptual trouble - how can the cell reap meaningful 1992). information from networks based on such a common module, Early studies indicated that the region bound by SH3 whose affinity and selectivity is modest at best? Because SH3 domains is in all cases proline-rich and identified PxxP as a domains play a role in so many of the processes of interest to core conserved binding motif (Ren et al., 1993). A host of the cell biology and biochemical communities, it is without subsequent studies using alanine-scanning mutagenesis of doubt important that we get a handle on what they can and known binding sites, phage display, combinatorial chemistry cannot do, how we can use their properties to our advantage and high-resolution structure determination have revealed the and how we can disrupt them in vivo. Here, I very briefly specifics of binding in great detail (recently reviewed by Kay sketch out the general properties of SH3 domains, spend et al., 2000). What is clear is that the surface of the SH3 domain considerable time on recent advances in understanding the bears a relatively flat, hydrophobic ligand-binding surface, determinants of binding specificity (or lack of specificity) and which consists of three shallow pockets or grooves defined by its significance, and go on to discuss two specific recent conserved aromatic residues (Fig. 1). The ligand adopts an examples in which these domains appear to play complex and extended, left-handed helical conformation termed the varied roles in important biological processes. polyproline-2 (or PPII) helix. The PPII helix has three residues per turn; this means it is roughly triangular in cross-section, and the base of this triangle sits on the surface of the SH3 A brief history domain. Two of the three ligand-binding pockets of the SH3 SH3 domains were first noted as regions of sequence similarity domain are occupied by two hydrophobic-proline (ΦP) between divergent signaling proteins such as the Src family of dipeptides in register on two adjacent turns of the helix, 1254 JOURNAL OF CELL SCIENCE 114 (7) A B C RT Class I ligand loop P P R Cα R N X X N Cα δ Cβ C X X n-Src loop C Class I N Class II ligand RT loop Cα X X R N X X R N Cα Cδ Cβ P P n-Src loop N Class II C Fig. 1. Interaction of SH3 domains with their ligands. (A) Binding of class I (top) and class II (bottom) ligands to the surface of the SH3 domain is depicted diagrammatically. The specificity pocket that typically interacts with an arginine residue in the ligand is to the right, the two Φ-P pockets to the left. The orientation of the ligand is indicated beneath each diagram. Approximate positions of the variable RT and N-Src loops of the SH3 domain are indicated. (B) The Src SH3 domain bound to class I (top) and class II (bottom) ligands (adapted from Feng et al., 1994). The surface of the SH3 domain was rendered with the program GRASP; ligands are represented in stick format. The overall view corresponds to the diagram in A. (C) Schematic view of an X-P dipeptide of a class I (top) or class II (bottom) ligand on the surface of an SH3 domain (adapted from Nguyen et al., 1998). The view is down the axis of the PPII helix of the ligand from the perspective of the specificity pocket of the SH3 domain. The surface of the SH3 domain is represented in green, and the portions of the X-P dipeptide that contact the surface are represented in red. Note that the majority of the proline ring is not in contact with the surface of the SH3 domain and that it is the Cδ atom of the proline ring (the atom directly bound to the main-chain nitrogen atom) that is in closest contact with the SH3 surface: N-substitution is thus the main feature recognized by SH3 domains. Note that in either orientation the Cα atom of the proline is directed away from the SH3 surface and does not contribute to the binding energy. whereas the third ‘specificity’ pocket in most cases interacts there is solid evidence for specific biologically significant SH3- with a basic residue in the ligand distal to the ΦPxΦP core. ligand interactions. The first and most prominent example was Remarkably, different binding modules such as WW domains the role of Grb2 (or Sem-5 in C. elegans and DRK in and profilin have converged on very similar modes of Drosophila) in activating Ras. Grb2 contains an SH2 domain interaction with proline-rich, PPII-helical ligands (Kay et al., that binds to tyrosine-phosphorylated proteins, which is 2000; Zarrinpar and Lim, 2000). flanked by two SH3 domains that bind to the proline-rich tail As discussed below, the affinity of SH3 domains for their of Sos (as well as other proteins). Sos is a guanine-nucleotide- peptide ligands is quite low, and selectivity is generally exchange factor (GEF) for the small GTPase Ras. Genetic and marginal at best; so it is reassuring that in a number of cases biochemical evidence demonstrated that Grb2 recruits Sos to SH3 domains: complexity in moderation 1255 tyrosine-phosphorylated sites on the membrane, where Ras is amide nitrogen of the proline residue, and the carbonyl, α localized by virtue of its covalent lipid modification, and that carbon and side chain of the preceding residue (Fig. 1c) Grb2 thereby couples activation of Ras to changes in tyrosine (Nguyen et al., 1998). An x-P dipeptide is the only possible phosphorylation (McCormick, 1993). While this remains the sequence of naturally occurring amino acids in which two paradigm for SH3-mediated intermolecular interactions, there alkylated backbone atoms are separated by only a single are now several other examples for which there is solid carbon. evidence, such as recruitment of the Pak kinase by the Dock Given that almost all of the binding energy is provided by SH2-SH3 adaptor during retinal axon migration in Drosophila the two ΦP dipeptides and the basic residue, there wouldn’t (Hing et al., 1999). seem to be much room for selectivity among different SH3 The most prominent example of the importance of domains. Despite this, several groups have used phage display intramolecular SH3-mediated interactions comes from Src or combinatorial chemistry approaches to identify optimal itself. Src and its relatives are normally held in an inactive ligands for particular SH3 domains and in general have been conformation, in which a C-terminal phosphotyrosine engages able to find consensus sequences showing some specificity for in an intramolecular interaction with the SH2 domain of the a particular domain (Cestra et al., 1999; Cheadle et al., 1994; kinase.