Crystal Structure and NMR Studies of the Apo SH2 Domains of ZAP-70:  Two Bikes Rather than a Tandem

Abstract

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

“…In line with this argument and considering that we use a large excess of bait protein in our domain-based pull-down we do not enrich any of the other SH2 domain containing proteins when compared with previous phosphopeptide enrichments (18,22). Phosphorylated Y571 is predominantly recognized by the N-terminal SH2 (N-SH2) domain of ZAP70 and the residues displaying the largest signal reduction in our NMR studies (R17, L40) are identical to those that are line-broadened when ZAP70-tSH2 binds to the singly phosphorylated ITAM peptides pYNEL or pYDVL (37). The core of the ADAP sequence pY 571 DSL is very similar to a single ITAM motif, and residues R17 and L40 directly interact with the phosphate group of the ligand (54).…”

Binding of a Diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: A hydrogen exchange mass spectrometry study

“…To°summarize,°in°Figure°8°the°percentage°of°total exchangeable hydrogens exchanged after 25 min D 2 O incubation°are°depicted°for°both°the°ligand-free°( Figure 8a)°and°the°bound°Syk°tSH2°(Figure°8b).°Exchange protection upon binding in some regions of the SH2 domains can be readily explained in terms of peptide shielding: these regions are directly involved in the protein-peptide binding site(s). Although no structure of ligand-free Syk tSH2 is available to date, a good overview of the protein dynamics can be obtained from its°family-related°Zap-70°(Figure°1b)°kinase.°In°the°NMR and crystallographic study of the tSH2 domains of the latter°protein° [18],°it°is°clearly°shown°that°the°almost complete length of the inter-SH2 domain is very dynamic.°Similarly,°in°the°Syk°tSH2°protein°(Figure°8a), the most solvent accessible regions (red) are encountered in the inter-SH2 linker region and C-terminus. The easily accessible protein fragments localized in the inter-SH2 domain region undergo an extensive reduction°in°solvent°accessibility°upon°ITAM°binding°( Figure 8b),°which°we°propose°is°attributable°to°a°dramatic increase in structural ordering or reduction in backbone dynamics by hydrogen bonding.…”

“…Zap-70 kinase has been revealed by X-ray crystallography, and its backbone dynamics has been studied by NMR (Figure 1b) [18]. Although Zap-70 and Syk do not share that much sequence homology (i.e., 55%) the overall topology of the Syk tSH2 domain is quite similar to the one in Zap-70 [17].…”

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…[6,10] This is supported by the structures of tSH2 in the bound and the free states of the other member of the Syk/ZAP family kinases: ZAP-70. [11,12] In the unbound state, the two SH2 domains of ZAP-70 are far apart from each other. Upon ITAM binding, the tSH2 of ZAP-70 undergoes a large conformational change, becoming locked in a more "closed" conformation.…”

“…Upon ITAM binding, the tSH2 of ZAP-70 undergoes a large conformational change, becoming locked in a more "closed" conformation. [12] For Syk tSH2 only the crystal structure of its bound form with a doubly phosphorylated ITAM is available ( Figure 1). The asymmetric unit contains six copies of the bound tSH2 with variable distances between the pY binding sites of the SH2 domains, indicating that a certain degree of flexibility also exists in the bound state.…”