NMR Spectroscopic Analysis of the Regulatory Module of ZAP-70

S, Sunitha Kumari V (2016) NMR Spectroscopic Analysis of the Regulatory Module of ZAP-70. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Sunitha Kumari V S (11MS027)) Sunitha_11MS027_MS_thesis.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

The Zeta Associated Protein kinase (ZAP-70) plays an important role in the development and the activation of T cells. ZAP-70 is a non-receptor tyrosine kinase which is activated by localizing to the membrane. ZAP-70 relocates to the membrane by binding to the doubly phosphorylated ITAM (ITAM²P) peptides located in the T cell receptor complex. The binding to the ITAM²P is mediated through the tandem SH2 (tSH2) domain. Large structural reorientation of two SH2 domains occurs upon ITAM²P binding, leading to releases of autoinhibition of the kinase domain. How does tSH2 Domain recognizes an ITAM²P motif and specificity of ITAM²P binding are not clearly understood. Recently Visperas P et al., 2015 reported that, a covalent modification of compound A at C119, a distal site from ITAM²P binding site in the interdomain A of tSH2 leads to the decreased ITAM²P binding affinity. Similar effect was observed when tSH2 domain was treated with hydrogen peroxide. The crystal structure of compound A modified tSH2 formed a close conformation that resembles an ITAM²P bound state. We hypothesize that a complex circuitry of intermolecular network at the tSH2 domain regulates the activity of the ZAP-70. In this study, we are investigating the regulatory mechanism of tSH2 domain of ZAP-70 by using NMR spectroscopy. We assigned the backbone resonances of an ITAM²P bound cysteine mutant (C117S and C256S) of tSH2 domain. We analyzed the secondary structure and backbone dynamics from analysis of 2D and 3D spectra and compared with that of ITAM²P bound wild type tSH2 domain. The secondary chemical shifts of some regions of SS mutant are found to be different compared to the calculated chemical shifts from the crystal structure of wild type tSH2. Interestingly, in the folded structure the residues come close together and form specific interactions. F117 from C of interdomain A and W235 from B helix of C-SH2 form an aromatic interaction in the ITAM²P bound crystal structure, however this interaction break in the apo state. Also, W165 from BC loop of C-SH2 domain form π-CH2 interaction with W235 and R121 form a cation- interaction with F117. Analysis of peak intensities indicates that, the region formed by the helices αC and αD from interdomain A and B helix and BC loop from C-SH2 domain might play an allosteric regulatory role of ITAM²P binding and kinase activation. The wild type spectrum was found to be more dynamic than the SS mutant tSH2. It clearly suggests that, the modification near to the proposed allosteric site is responsible for the difference in the dynamics. From the present study, we hypothesize that the dynamics of the region formed by the helices C and D from interdomain A and αB helix and BC loop from C-SH2 domain control the ITAM²P binding kinetics and ZAP-70 activation.

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