Structure, function and engineering of peptide -MHC binding receptors

The goal of this work is to determine the molecular basis of specificity of the two major class I MHC binding proteins, T cell receptors (TCRs) and natural killer receptors (NKRs). We have shown previously that affinity maturation of the 2C TCR (complementarity determining region 3alpha, CDR3alpha) can be used to study peptide MHC cross-reactivity and kinetics. Here we employ a similar strategy to examine the NK receptor Ly49C and the other CDRs of the 2C TCR.;In chapter 2, the engineering of the inhibitory NK receptor Ly49C by yeast-display is described. Affinity-maturation of Ly49C enabled crystallization of the mutant Ly49C:OVA/Kb complex by our collaborators Julie Dam and Roy Mariuzza, revealed the stoichiometry of this interaction, and allowed the more detailed functional and biochemical analyses described in chapters 3 and 4.;Chapter 3 focuses on the use of affinity-matured Ly49C variants to study various aspects of its interaction with peptide-MHC. Higher affinity variants exhibited an increase in cis-interactions with host cell MHC molecules, possibly explaining why the affinity of NK receptors have evolved to be low (i.e. to minimize such cis interactions). Nevertheless, the Ly49C variants maintained peptide selectivity and revealed there is only a several fold difference in affinities that accounts for functional and non-functional ligands. The molecular basis of beta2m species specificity was determined using several key Ly49C mutants.;In chapter 4, the functional aspects of Ly49C were examined in more detail. A co-transfection system was used in which the 2C TCR served as the activating receptor. Three different forms of ligand presentation for the TCR and NKR were explored: (1) cis-presentation in which ligands for the TCR and NKR were both immobilized on plate, (2) trans -presentation in which the TCR ligand was oriented on a surface and the NKR ligand was present on an antigen presenting cell, and (3) shared ligand presentation in which the ligand for both the TCR and NKR was the same and presented on an antigen presenting cell. The results revealed a strong requirement for proximity of the ligands for the inhibitory and activating receptors in order to achieve efficient cellular inhibition. The finding is discussed in terms of a model of NK receptor function in memory T cells.;In chapter 5, a T cell receptor interaction with its peptide-MHC ligand was explored using various TCR mutants engineered by yeast display. Previous work in the lab showed that mutations in the CDR3 regions of the TCR could generate higher-affinity, peptide-specific TCR variants. In contrast, other CDRs (CDR1, and especially CDR2) of the TCR are typically located over the MHC helices and thus may not be positioned to interact directly with peptide. Mutations in these CDRs might be expected to yield high-affinity but reduced peptide specificity (i.e. interactions with MHC determinants). Surprisingly, mutants in CDR1 and CDR2 retained peptide specificity, suggesting that even TCR regions that are not directly in contact with the peptide can influence peptide specificity. Furthermore, random mutagenesis of the TCR revealed that single-site mutations are sufficient to achieve higher-affinities. One mutation in a residue (Valpha1) outside of the CDRs, also generated a higher affinity, peptide specific TCR. Results with this and other mutants led to various predictions about the plasticity of TCRs in recognizing specific antigenic pepMHC complexes.