Investigation of the Regulatory Mechanism of the ZAP-70 Immunological Signaling Enzyme

Abstract

ZAP-70 is a critical Syk-family protein tyrosine kinase (PTK) that functions in the initial step of T-cell receptor (TCR) signaling. Its importance is highlighted by mutations in ZAP-70 that cause severe combined immunodeficiency (SCID). Moreover, elevated levels of ZAP-70 are associated with T-cell proliferative diseases and chronic lymphocytic leukemia (CLL). Under normal conditions, T-cell activation by foreign antigen results in co-localization with ZAP-70, due to binding of its associated tandem SH2 domains (tSH2) to phosphorylated ITAM (Immunoreceptor Tyrosine Activation Motif) sequences present on the TCR ζ subunits. This leads to up-regulation of kinase activity and downstream initiation of an immune response. Experimental structures reveal that upon ITAM binding, the tSH2 must undergo a large conformational change. Of interest, therefore, is how ITAM-binding controls the tSH2 conformational equilibrium, and the subsequent proposed disassembly of the kinase domain. To this end, we have carried out 1.5 μs of molecular dynamics (MD) simulations of various ZAP-70 systems, composed of 50,000 to 70,000 atoms. These include the isolated tSH2, and variants of the ITAM-bound state. Collectively, our results suggest that one phosphotyrosine site regulates the tSH2 conformational equilibrium, whilst the other controls localization of the tSH2 to membrane-proximal ITAM targets. Using these results, along with those from targeted MD simulations, we have identified a set of order parameters that describe the tSH2 conformational transition. We have subsequently performed multidimensional potential of mean force (PMF) calculations, via umbrella-sampling, to describe the transition; a single such PMF for the tSH2 “switch” constitutes over 3 μs of simulation time. Thus, we have achieved an atomic level characterization of the free-energy landscape of the tSH2 in the presence and absence of the catalytic domain, with important consequences for the mechanism of signaling and autoinhibition in Syk-family kinases.