Understanding the Role of Allosteric Network in the tandem SH2 domain of ZAP70 and Syk Regulation

Debnath, Olivia (2018) Understanding the Role of Allosteric Network in the tandem SH2 domain of ZAP70 and Syk Regulation. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Olivia Debnath (13MS049)) 13MS049.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

The T-Cell signaling initiates with the binding of Antigen Presenting Cell with T-cell Receptor (TCR) Complex leading to the recruitment of a non-receptor tyrosine kinase, namely Zeta Associated Protein-70 (ZAP-70), to the membrane. ZAP-70 consists of a regulatory module called as tandem SH2 domain (t-SH2) comprising of N-SH2 and C-SH2 domains, and a carboxy-terminal kinase domain [70]. N-SH2 and C-SH2 are connected by coiled-coil like Interdomain-A whereas Interdomain-B connects tSH2 domain with the kinase domain. In cytoplasm, ZAP70 remains in an autoinhibited state and upon binding to the doubly-phosphorylated Immunoreceptor Tyrosine Activation Motif (pY-ITAM) peptide in TCR complex, the ZAP-70 is recruited to the membrane [19,20]. The ITAM motif has two phosphotyrosines (pY) that cooperatively binds to ZAP70 t-SH2 domain. This binding causes a conformational rearrangement of the regulatory module, thereby releasing the autoinhibitory conformation of the kinase domain of ZAP70. However, in its counterpart in B-cell signaling; Syk, the t-SH2 domain binds to the doubly phosphorylated peptide in a non-cooperative fashion. Despite having high structural and 57% sequence similarity, the mechanistic detail of why the two tSH2 domains of ZAP-70 and Syk show distinct binding patterns towards pY-ITAM is not understood. To investigate the fundamental differences between these two proteins, I have employed structural bioinformatics approach- coarse grained residue interaction network analysis and biochemical assays. Residue interaction network (RIN) analysis of the ZAP70 t-SH2 domain bound to the peptide revealed an allosteric network involving F117-W235 π- π aromatic stacking that couples the two SH2 domains. Also, the identified residues involved in the ITAM binding network acts as important hubs and are evolutionarily conserved. This information network mediated by the allosteric nodal hub is perturbed in the apo state of the protein. The RIN obtained for pY-ITAM bound Syk does not involve the allosteric hub and the shortest RIN involving ITAM binding in Syk can even proceed through residues which are absent in the ZAP70 network. Experimental evidences from our lab supports that mutating these nodal hub residues perturb the ITAM binding to ZAP70. To understand, whether these nodal residues mediating allostery in ZAP70 has any importance in Syk kinase, a site directed mutagenesis was performed on Syk to generate F117A mutant (Phenylalanine mutated to Alanine). Subsequently, upon conducting thermal denaturation by circular dichroism and intrinsic fluorescence assay, it was found that this mutation neither destabilize the Syk structure nor affects its ITAM binding. Therefore, the information network involving F117-W235 π-π aromatic stacking might be an incredible feature that can distinguish binding patterns of ZAP70 and Syk towards pY-ITAM- which means, this ‘allosteric switch’ might be very crucial for the allosteric regulation of ZAP70; whereas the absence of it in the information network may cause non-cooperative ITAM binding in Syk.

Actions (login required)

View Item