title: 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.
authors.affiliation: Structural Chemistry Laboratory, AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden.
authors.forename: Rutger H A
authors.initials: RH
authors.lastname: Folmer
authors.forename: Stefan
authors.initials: S
authors.lastname: Geschwindner
authors.forename: Yafeng
authors.initials: Y
authors.lastname: Xue
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry; D001665:Binding Sites; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019000:Phosphotyrosine / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
chemical_list: D001051:Apoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase