The Src homology 2 (SH2) domain has a special role as one of the cornerstone examples of a “modular” domain. The interactions of this domain are very well-conserved, and have long been described as a bidentate, or “two-pronged plug” interaction between the domain and a phosphotyrosine (pTyr) peptide. Recent work has, however, highlighted unusual features of the SH2 domain that illustrate a greater diversity than was previously appreciated. In this review we discuss some of the novel and unusual characteristics across the SH2 family, including unusual peptide binding pockets, multiple pTyr recognition sites, recognition sites for unphosphorylated peptides, and recently identified variability in the conserved FLVR motif.
In 1986, a new domain was identified within v-src, the transforming gene of the Rous sarcoma virus. This region had high sequence similarity across the then-known cytoplasmic protein-tyrosine kinases, and a conserved location N-terminal to the kinase domain (the first homology region identified in Src). The domain was termed the Src homology 2 (SH2) domain, and its identification heralded a new era in the understanding of molecular interactions and cellular signaling. SH2 domains have been critical for development of key concepts such as the dependency of cytosolic signaling on post-translational modification-regulated protein interactions, and the modularity of protein domains. Over the years, extremely well conserved molecular mechanisms have been revealed which are used by SH2 domains to mediate their effects. These canonical features are well-documented, but unusual features also occur and increase the diversity of the fold. In this review, we discuss these unusual features and how they exhibit divergence from canonical SH2 domain architecture.
The primary molecular role of the SH2 domain is to directly bind phosphotyrosine (pTyr) residues. This is central to propagation of signaling by receptor and non-receptor tyrosine kinases such as the insulin receptor and the JAK kinases, so SH2 domains are critical to a range of fields including endocrinology. The SH2-pTyr interaction is broadly independent of folding of the pTyr-ligand, and can be observed for denatured Tyr peptides, but is distinct from recognition of pTyr by for example the phosphotyrosine binding (PTB) domains. Thus, the binding of SH2 domains to short linear peptide motifs can be predictive for the interactome of specific SH2 domains. SH2 interaction selectivity has yielded extensive knowledge of their binding partner preferences and signaling networks.
The mechanisms for SH2-ligand interaction are well-defined, with the first cohort of structures for this ~100 amino acid fold determined in 1992 and 1993. These structures showed that the SH2 domain consists of a central β-sheet flanked by two α-helices. They revealed that the phosphorylated peptide binds perpendicularly to the β-sheet and docks into two abutting recognition sites formed by the β-sheet with each of the α-helices. This bidentate, or “two-pronged plug”, interaction provides both a deep basic pTyr binding site, and a specificity pocket that usually recognizes an amino acid three residues C-terminal to the pTyr (termed the +3 position), a mode of interaction that is consistent in vitro and in cells. The nomenclature for the fold defines the antiparallel β-strands as βA-βG and the helices as αA and αB, with loops named by the flanking secondary structure. Thus, the pTyr pocket is canonically defined by residues of αA, βB, βC, βD, and by the BC “phosphate binding loop”; and the specificity pocket by residues of αB, βG, and the BG and EF loops.
Evolutionarily, the SH2 domains appear early in the eukaryotic phylogenetic tree and are thought to have co-evolved with tyrosine kinases to the complex array of pTyr-responsive signaling is found in humans. Indeed, an ancestral SH2 domain appears to have been identified in SPT6, a transcription elongation factor universally present from yeast to humans. As discussed below, SPT6 maintains the overall SH2 fold but binds to phospho-serine and phospho-threonine thus providing a stepping stone to pTyr binding. Unusual SH2 domains have also been acquired and evolved by some bacteria, presumably for invasive purposes, and below we discuss some of those contained in the Legionella genome.
Within the canonically defined pTyr pocket there are conserved residue motifs. The most critical motif for pTyr binding includes an arginine at the fifth position of βB, βB5, as part of a highly conserved “FLVR” or “FLVRES” amino acid motif. As much as half of the free energy of binding is lost on point mutation of this residue resulting in a 1,000-fold reduction in binding affinity, and it provides a floor at the base of the deep pTyr pocket which allows specificity toward pTyr over pSer/pThr. The FLVR arginine residue is considered key to pTyr recognition, and is conserved in all but 3 of the 120+ human SH2 domains.
Other conserved residues which often work in concert with βB5 to coordinate pTyr have been identified. The most prominent are basic residues (arginine or lysine) at positions αA2 and βD6. Coordination of pTyr by both αA2 and βD6 is rarely observed in the same SH2 domain and this observation has allowed the definition of two major classes, the Src-like (with a basic residue at αA2), and the SAP-like (with a basic residue at βD6) SH2 domains, referencing two of the most well studied members of the family. Experimentally, however, Arg βB5 is the residue most often targeted by point mutagenesis to interrupt SH2-pTyr binding.
Outside of the canonical binding site, which provides recognition of the pTyr and +3 positions, interfaces have been found to contribute to binding at a range extending to the −6 and +6 positions. Larger interaction and alternative surfaces have also been observed, for example to achieve high FGFR1 selectivity the N-terminal SH2 domain of PLCγ1 uses an extended surface, but its C-terminal SH2 domain does not and is consequently a weaker binder. An alternate surface is also used by the SAP SH2 domain which interacts with the SH3 domain of Fyn using a region distal to its pTyr binding site. Despite these findings, however, the foundational conserved mode of binding for most SH2 domains to pTyr ligands is centered on the interactions of pTyr and the +3 position. Nonetheless, over the course of study of SH2 domains exceptions to these general rules have been observed.
The exceptions to the canonical “two-pronged plug” binding observed thus far create a diversity in the recognition patterns by which SH2 domains can bind their partners. These exceptions include unusual binding pockets, unique specificities, and dependency of oligomeric state for binding. Below, we highlight some of the mechanisms SH2 domains use to select binding partners and discuss diversity within the pTyr binding site, starting with ancestral SH2 domains before proceeding to eukaryotic SH2 domains.
Probably the most ancient SH2 domain discovered to date is found in SPT6, an essential transcription elongation protein. This protein contains tandem SH2 domains which are the only two SH2 domains in yeast. They pack against one another and recognize extended phosphorylated serine and threonine peptides of RNA polymerase II. The C-terminal SH2 domain lacks a canonical phospho-binding site, but instead has a pocket on its back side which binds a pSer in its binding partner. In contrast, the N-terminal SH2 domain has a near canonical phospho-binding pocket which recognizes pThr, and its recent structure-guided analysis showed that the N-SH2 pocket preferentially binds pThr followed by Tyr. This pT-X-Y motif makes use of the FLVR arginine to coordinate the pThr's phosphate, but the Tyr is also oriented into this pocket in a manner similar to the aromatic region of a canonical pTyr-SH2 interaction. The coordination of both pThr and Tyr by SPT6 therefore resembles a canonical pTyr-SH2 interaction, making this potentially the evolutionary stepping-stone to SH2-media
