MODELLING OF ZAP-70 ACTIVATION UPON PHOSPHORYLATION OF TYR315, TYR319 AND TYR493 FOR SIGNALLING T-CELL ACTIVATION

Abstract

<h3>Introduction</h3> ZAP–70 is a key kinase in the regulation of the adaptive immune response. Zap-70 acts by binding its SH2-domains to T-cell-associated CD3ζ protein, thus transmitting T-cell activation signals induced by the interaction of Major Histocompatibility Complexes with T-cell Receptors. Phosphorylation of Tyr315, Tyr319 and Tyr 493 is required for ZAP-70 kinase activation by unclear mechanisms. In this study tools of structural bioinformatics were used to elucidate the mechanisms of ZAP-70 activation. <h3>Methods</h3> The consequences of the phosphorylation of Tyr315, Tyr319, Tyr493 were studied using 300 ns Molecular Dynamics simulations with GROMACS software and GROMOS 54a7 forcefield allowing assessment of phosphorylated tyrosines. <h3>Results</h3> The atomic structure of full-length nonphosphorylated ZAP-70 was first predicted to be next used in MD-simulations. The simulations show that phosphorylation of Tyr315, Tyr319, Tyr493 stabilizes ZAP-70 activation loop through the formation of hydrogen bonds of pTyr493 with Asp489 and Asp490, as well as through salt bridge formation between pTyr493 and Arg460. These results suggest that this stabilization of the ZAP-70 kinase domain activation loop allows the subsequent phosphorylation of LAT and SLP-76 by ZAP-70. <h3>Conclusion</h3> According to this scenario, the role of phosphorylation of Tyr315 and Tyr319 is in the separation of N-SH2 and C-SH2 domains through distance providing space complementarity between ZAP-70 and CD3ζ dimer ITAM-motives which results in ZAP-70 recruitment to T-cell membranes where phosphorylation of Tyr493 by Lck-kinase occurs.