22T-cell receptor (TCR) signaling is initiated by recruiting ZAP-70 to the cytosolic part of TCR. ZAP-70, a 23 non-receptor tyrosine kinase, is composed of an N-terminal tandem SH2 (tSH2) domain connected to 24 the C-terminal kinase domain. The ZAP-70 is recruited to the membrane through binding of tSH2 25 domain and the doubly-phosphorylated ITAM motifs of CD3 chains in the TCR complex. Our results
26show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-27 phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine 28 residue of ITAM peptide to form an encounter complex leading to subsequent binding of second 29 phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that 30 allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in 31 the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to 32 the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma 33 membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is 34 unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in 35 B-cells.
36Significance 37 38 T-cell and B-cell signaling is initiated by the same family of non-receptor tyrosine kinases, ZAP-70 and 39 Syk, respectively. ZAP-70 and Syk share homologous sequence and similar structural architecture, yet 40 the two kinases differ in their mode of ligand recognition. ZAP-70 binds cooperatively to its ligand,
41whereas Syk binds uncooperatively. Spontaneous mutation (W165C) in the regulatory module of ZAP-42 70 impairs T-cell signaling causes autoimmune arthritis in SKG mice, the mechanism of which is 43 unknown. We showed that ZAP-70 regulatory module undergoes a biphasic structural transition while 44 binding to its ligand, which is fundamentally different from Syk. We presented a molecular mechanism 45 of cooperativity in ZAP-70 regulatory module that explains altered ligand binding by ZAP-70 mutant 46 found in SKG mice. 47 48 49 50 51The zeta-chain-associated protein tyrosine kinase, ZAP-70, is a non-receptor tyrosine kinase 52 crucial for T-cell signaling, development, activation, and proliferation(1-4). T-cell signaling is 53 commenced by the recruitment of two protein tyrosine kinase, Src family kinase Lck and ZAP-70, to the 54 activated molecular complex of T-cell antigen receptor (TCR)(5, 6). Lck, phosphorylate several tyrosine 55 residues of the immuno-receptor tyrosine-based activation motifs (ITAM) on the intracellular segment 56 of CD3 heterodimer (made up of d, g, and e) and ζ homodimer associated with the TCR(5, 7-10). ZAP-57 70 is spontaneously recruited to the membrane by binding to the doubly-phosphorylated ITAM (ITAM-58 Y2P) motifs (11)(12)(13)(14). Recruitment of ZAP-70 allows phosphorylation of scaffold proteins that initiates a 59 cascade of downstream biological events (15, 16)...