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

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 TCRζDP1 (L51-D93) polypeptide grew in a tetragonal space group but only diffracted to low resolution. In contrast, crystals of the Nefcore-TCRζA63-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 Nefcore-TCRζA63-R80 complex was determined using refinement procedures that accounted for crystal twinning to 2.05 Å. The structure of the Nefcore-TCRζDP1 complex was solved to 3.7 Å from a single non-twinned crystal. The altered crystal packing induced by the shorter TCRζA63-R80polypeptide is postulated to have led to a reduction in crystal symmetry and increase in proneness to crystal twinning. Chapter IV provides a detailed analysis of the structure of the Nefcore-TCRζA63-R80 complex and demonstrates its effect on modulation of Src family kinase activity. The TCRζ polypeptide adopts an alpha helical conformation and occupies a hydrophobic crevice on Nef not shared by any of Nef’s reported interaction partners. The interaction of Nefcore with TCRζ is mediated primarily by the burial of hydrophobic residues on TCRζ (L75, L77) in a hydrophobic pocket on Nef and a salt bridge between a glutamic acid (E74) on TCRζ and a basic patch on Nef consisting of two conserved arginines (R105, R106). The TCRζ polypeptide additionally orders the N-terminus of Nefcore into a polyproline type II helix that has been described to bind the SH3 domain of Src family kinases. We demonstrate that in vitro phosphorylation of TCRζcyt by Fyn and Src is specifically augmented by HIV-1 and SIV Nefcoreand suggest that Nef-TCRζ complex formation cooperatively enhances kinase activity. Chapter V contains overall conclusions, future directions and a model illustrating the proposed role of the Nef-TCRζ interaction in immune activation modulation. The Appendices contain sequences of the proteins, gene constructs and primers used in this work

Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRzeta fragments.

HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling zeta subunit of the T-cell receptor (TCRzeta). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nef(core)) in complex with two different TCRzeta fragments are described. The structure of SIVmac239 Nef(core) bound to the longer TCRzeta polypeptide (Leu51-Asp93) was determined to 3.7 A resolution (R(work) = 28.7%) in the tetragonal space group P4(3)2(1)2. The structure of SIVmac239 Nef(core) in complex with the shorter TCRzeta polypeptide (Ala63-Arg80) was determined to 2.05 A resolution (R(work) = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P2(1)2(1)2(1). The reduction in crystal space-group symmetry induced by the truncated TCRzeta polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, -l) and a and b unit-cell parameters that were nearly identical predisposed the P2(1)2(1)2(1) crystal form to pseudo-merohedral twinning

The intrinsically disordered cytoplasmic domain of the T cell receptor zeta chain binds to the nef protein of simian immunodeficiency virus without a disorder-to-order transition.

Intrinsically disordered proteins are thought to undergo coupled binding and folding upon interaction with their folded partners. In this study, we investigate whether binding of the intrinsically disordered T cell receptor zeta cytoplasmic tail to the well-folded simian immunodeficiency virus Nef core domain is accompanied by a disorder-to-order transition. We show that zeta forms a 1:1 complex with Nef and remains unfolded in the complex. Thus, our findings oppose the generally accepted view of the behavior of intrinsically disordered proteins and provide new evidence of the existence of specific interactions for unfolded protein molecules