Structure of SH2 Domains

SH2 domains generally consist of approximately 100 amino acid residues [49,50, 60,61 ] and have been first identified as a conserved sequence region between the oncoproteins Src and Fps [19, 48]. By means of sequence homology, SH2 domains have been uncovered in numerous other intracellular signal transduction proteins (Figs. 1 and 4) [20]. 

Due to the ready accessibility of SH2 domains by molecular biology techniques, numerous experimentally determined 3D structures of SH2 domains derived by X-ray crystallography as well as heteronuclear multidimensional NMR spectroscopy are known today. The current version of the protein structure database, accessible to the scientific community by, e.g., the Internet (http //www.rcsb.org/pdb/) contains around 80 entries of SH2 domain structures and complexes thereof. Today, the SH2 domain structures of Hck [62], Src [63-66], Abl [67], Grb2 [68-71], Syp [72], PLCy [73], Fyn [74], SAP [75], Lck [76,77], the C- and N-terminal SH2 domain ofp85a [78-80], and of the tandem SH2 domains Syk [81,82], ZAP70 [83,84], and SHP-2 [85] are determined. All SH2 domains display a conserved 3D structure as can be expected from multiple sequence alignments (Fig. 4). The common structural fold consists of a central three-stranded antiparallel ft sheet that is occasionally extended by one to three additional short strands (Fig. 5). This central ft sheet forms the spine of the domain which is flanked on both sides by regular a helices [49, 50,60]. 

GsIfsL SVRDFDqNlqG Ettk.GaycL SVsDFDNaKG dnn..GsyaL cll.heG 

LDnGGfYISp LDsGGfYIts dktGklsIpe. DGaGkYflw. tGdyydlyg. rdGkygfSd LDGGG-YIS- 

VrhYtNa sDG VayYSkP aDG VehYSylaDG VdyHrst svs VqyYmePhgq inhYrNe sla V—YSNE-DG 

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The structure of SH2 domains can be conveniendy divided into two functional regions that involved in coordination of the pTyr of the target and that which contacts the residues G-terminal to the pTyr (Fig. 2A). The pTyr binding cavity is found in the N-terminal half of the protein between the central p-sheet and helix oA, while the specificity determining region is found primarily in the C-terminal half of the protein between the central p-sheet and hehx aB (Fig. 2A). Each of these regions is described in detail below. 

Fig. 2. Structures of SH2 domains. (A) Stereo ribbon diagram of an SH2 domain. The structure shown is the SH2 domain of the Src kinase complexed with the high-...

The structures of SH2 domains in the context of other protein modules provided much information on how these modules function within the context of a larger protein. However, the availibility of structures is just the first step of many which are needed to understand how macromolecules function. In this section, we describe solution studies of SH2 domains placed in the context of either larger protein fragments or full-length proteins that have provided information about the various mechanisms by which SH2 domains regidate protein function. 

This contribution will summarize the currently pursued approaches towards non-peptide SH2 domain antagonists and the development of promising lead structures for SH2 domain-associated disease processes. It is clearly beyond the scope of this review to provide an in-depth overview of the entire research area. 

The crystal structures of the SH2 domains of Src tyrosine kinase and Lck tyrosine kinase in complex with Tyr phosphorylated peptides have enabled important insight to be obtained into recognition of the phosphotyrosine residue and the neighboring amino acids in class lA of SH2 domains. The phosphate residue is boimd in a deep pocket of the SH2 domain, at the end of which an invariant Arg residue (Arg PB5) is located which contacts the negatively charged phosphate by a two-pronged interaction. It can be estimated that a phosphoserine or phosphothreonine residue would be too short to enter into a similar interaction with the Arg residue. 

Cytoplasmically localized protein tyrosine phosphatases have a catalytic domain and other structural elements that specify the subcellular localization and association with effector molecules. These structural elements contain sequence signals for nuclear localization, for membrane association and for association with the cytoskeleton (see Fig. 8.16). The presence of SH2 domains suggests that these molecules might interact with signaling pathways involving growth hormones and receptor tyrosine kinases. 

Structural Insights, SH2 Domains An Example of Modular Regulatory Domains, takes a close look at phosphotyrosine-SH2 domain interactions and the diverse ways they can affect protein function. 

The first SH2 domain structures to be solved were those of the Src tyrosine kinase (Waksman et al., 1992), the Abl tyrosine kinase (Overduin et al., 1992), and the N-terminal SH2 domain of the p85 subunit of the PI 3 -kinase (N-p85 SH2 domain) (Booker et al., 1992). These structures revealed the architecture of SH2 domains. The SH2 domain fold is relatively simple it consists of a central antiparallel (3-sheet flanked by... 

While the interactions at the pTyr binding pocket are generally similar for all SH2 domains, those which involve residues other than pTyr are not. These interactions help to determine the specificity of SH2 domain-target recognition. Comparison of the many SH2 domain-phosphopeptide structures has revealed that different SH2 domains use somewhat different mechanisms to engage their respective targets. 

Information about the specificity of SH2 domain binding came initially from the structures of the Src and Lck SH2 domains in complex with tyrosyl phosphopeptides with the sequence Glu, Glu, He (EEI) C-ter-minal to the pTyr (pY) (Eck et al., 1993 Waksman et al., 1993). This pYEEI sequence had previously been identified to be specific for the Src family of SH2 domains, which includes Lck (see Section III). The Src and Lck structures are very similar to one another. Although the peptides employed in both studies contained residues outside the pYEEI motif, the structures indicate that only these four residues contact the SH2 domain. The peptide binds the SH2 domains perpendicular to the central p-sheet in an extended conformation (Fig. 2A). The pTyr makes contacts very similar to those observed for the structure of the Src SH2 domain in complex with low-affinity tyrosyl phosphopeptides (see above). However, in addition, the EEI motif makes specific contact with other residues of the protein. 

IV. Structure and Function of SH2 Domains in the Context of Other Protein Modules... 

Initial investigations of SH2 domains focused on understanding how these domains functioned in isolation. As described in Sections II and III, SH2 domains were expressed, purihed, and studied in vitro alone in order to understand their structure and mechanism of binding. WhUe this reductionist approach has allowed extensive characterization of SH2 domains, studies of isolated SH2 domains have not addressed how SH2 domains communicate with other protein domains in order to determine the biological function of SH2 domain-containing proteins. In this section, we describe both structural (Section IV,A) and solution-based biophysical (Section IV,B) investigations which have probed the mechanisms by which SH2 domains function within the context of other domains or of full-length proteins. 

Pawson and Scott, 1997). These modules are 60 amino acids in length and form a five-stranded 3-sandwhich structure Musacchio et al., 1992 Yu et al., 1992 see also the chapter hy Musacchio, this volume) their function is to hind proline-rich sequences in cellular proteins and hence facilitate the assembly of signaling complexes (Koch et al., 1991). A key question for the many proteins which contain hoth SH2 and SH3 domains is whether these domains cooperate in binding. Information regarding this question has been obtained from the structures of SH2-SH3 domain constructs, with the relevant data being the extent of molecular contacts and orientation between the SH2 and the SH3 domains. 

While the structures of SH2-SH3 domain constructs described above provide some hint at ways in which SH2 domains might interact with... 

Other domains, a better understanding of SH2 domain interactions requires examination of entire SH2 domain-containing proteins. In that respect, the structures of several Src family kinases have gready illuminated the mechanism by which the SH2 domain of these proteins communicates with the kinase domain in order to regulate enzyme activity. These structures were those for human Src (Xu et al., 1997), chicken Src (Williams et al., 1997), and the Src family member Hck (hematopoietic cell kinase) (Sicheri et al., 1997). 

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