Characterization of the Nef-TCR Zeta Interaction and Its Role in Modulation of Src Family Kinase Activity: A Dissertation

Abstract

One of the hallmarks of an infection with pathogenic HIV-1 is the elevated level of immune activation that leads to rapid progression to AIDS. Surprisingly, nonhuman primates naturally infected with SIV do not exhibit an augmented activation phenotype nor severe immunodeficiency. One of the viral components implicated in determining the state of immune activation is the accessory protein Nef which has been demonstrated to affect T cell signaling pathways from within the intracellular compartment and for Nef from SIV, to downregulate TCR surface expression. Recently, Nef from HIV-1 and SIV have been demonstrated to bind the ζ chain of the TCR which functions as the primary signaling subunit of the receptor. However, the molecular details of the Nef-TCRζ interaction as well as the role of complex formation in modulation of immune activation remain largely unknown. This thesis describes work directed at elucidating the biochemical and structural features of the Nef-TCRζ interaction and the functional consequences of complex formation relevant to T cell activation. Chapter I provides a brief introduction on HIV/SIV classification and pathogenesis with an emphasis on Nef and its pleiotropic function in T cells. Chapter II describes the biochemical characterization of the interaction of the conserved core domain of Nef proteins from HIV-1, HIV-2 and SIV with the cytoplasmic domain of TCRζ. The core domains of HIV-2 ST and SIVmac239 are demonstrated to bind the cytoplasmic domain of TCRζ at two distinct regions and with different affinities. In contrast, the core domain of HIV-1 isolate ELI Nef only binds to one region and with the weakest calculated affinity among the HIV-1, HIV-2 and SIV Nef proteins studied. In addition, both the N-terminal domain and the strong TCRζ-binding core domain of SIVmac239 Nef each are demonstrated to be necessary but not sufficient for downregulation of TCR surface expression. Chapter III describes the crystallization and structure determination methods used to solve the crystal structures of the core domain of SIVmac239 Nef in complex with two overlapping TCRζ polypeptides. Crystals of Nef in complex with the longer TCRζ DP1 (L51-D93) polypeptide grew in a tetragonal space group but only diffracted to low resolution. In contrast, crystals of the Nef core -TCRζ A63-R80 complex grew in an orthorhombic space group and diffracted to high resolution but were nearly perfectly pseudo-merohedrally twinned thus complicating structure determination. Following identification of the twin law relating the twin domains, the structure of the Nef core -TCRζ A63-R80 complex was determined using refinement procedures that accounted for crystal twinning to 2.05 A. The structure of the Nef core -TCRζ DP1 complex was solved to 3.7 A from a single non-twinned crystal. The altered crystal packing induced by the shorter TCRζ A63-R80 polypeptide is postulated to have led to a reduction in crystal symmetry and increase in…