Understanding the role of Zeta Associated Protein Kinase-70 (ZAP-70) tandem SH2 domain dynamics in T-cell signaling

Gangopadhyay, Kaustav (2022) Understanding the role of Zeta Associated Protein Kinase-70 (ZAP-70) tandem SH2 domain dynamics in T-cell signaling. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Kaustav Gangopadhyay (15RS048)) 15RS048_DBS.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

T cell signaling starts with assembling several tyrosine kinases and adaptor proteins to the T cell receptor (TCR), following the antigen binding. ZAP-70 is one of the non-receptor tyrosine kinases, which plays a significant role in the T-cell initiation complex. The protein is recruited to the ITAM-Y2P of the CD3ζ chain by the two SH2 domains (tSH2 domain), which helps in the activation of the kinase domain. But it is not clear which SH2 domain binds first, as well as how the two SH2 domains crosstalk. Our results show that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly-phosphorylated ITAM-ζ1 peptide. The C-terminal SH2 domain binds first to the phosphotyrosine residue of ITAM peptide to form an encounter complex leading to subsequent binding of second phosphotyrosine residue to the N-SH2 domain. We decipher a network of non-covalent interactions that allosterically couple the two SH2 domains during binding to doubly-phosphorylated ITAMs. Mutation in the allosteric network residues, for example, W165C, uncouples the formation of encounter complex to the subsequent ITAM binding thus explaining the altered recruitment of ZAP-70 to the plasma membrane causing autoimmune arthritis in mice. The proposed mechanism of allosteric coupling is unique to ZAP-70, which is fundamentally different from Syk, a close homolog of ZAP-70 expressed in B-cells.Hence, we plan to understand the functional significance of the different ITAM-Y2P binding by ZAP-70 and Syk. Previous studies suggested that the lifetime of the TCR: antigen complex and the time delay between the recruitment and activation of each kinase determines the T cell response. The mechanism by which the time delays are implemented in TCR signaling is not fully understood. Combining experiments and kinetic modeling, we here report a thermodynamic-brake in the regulatory module of ZAP-70, which determines the ligand selectivity and may delay the ZAP-70 activation in TCR. Weakening of the allosteric network led to decreased rate of conformational transition as well as higher thermodynamic penalty to form the closed state, suggesting that the allosteric network constitutes the thermodynamic brake which imparts a delay between ZAP-70: ITAM-Y2P binding and activation. Paralogous kinase Syk expressed in B cells does not possess such a functional thermodynamic brake, which may explain higher basal activation and lack of ligand selectivity by Syk.

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