Solution mapping of T cell receptor docking footprints on peptide-MHC

Abstract

T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a ''recognition code'' between conserved elements of the TCR and MHC genes. Although Ϸ17 cocrystal structures of unique TCRpMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2L d -QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a highaffinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many L d -specific TCRs.chemical shift mapping ͉ dynamics ͉ NMR ͉ cellular immunity ͉ protein-protein interaction T cells modulate the nature and extent of an immune response based primarily on specific T cell receptor (TCR) recognition of peptide in the context of a MHC molecule (1, 2). The TCR is a genetically recombined receptor, analogous to an antibody, that is composed of ␣ and ␤ chains. The binding site of the TCR comprises six loops called complementarity-determining regions (CDRs), with each chain contributing three loops, called CDR 1, 2, and 3. The CDR1 and CDR2 loop sequences are encoded by the variable (V) genes, but because there is no somatic mutation in TCR-V genes, these are referred to as ''germ line-derived.'' In contrast, the CDR3 loops, which are largely involved in antigenic peptide contact and thus specificity, are derived from recombination of V(D)J segments and vary in an almost unlimited fashion. MHC gene products present antigenic peptides to the TCR through a composite peptide-MHC (pMHC) surface composed of a highly variable element (i.e., peptide) in a groove surrounded by more conserved residues on the MHC helices (2).The interaction between TCR and pMHC has been studied through cocrystallization of Ϸ17 unique complexes (1, 3). Some general principles have emerged from the current database of complexes (4). Briefly, the TCR has an orientation over the pMHC surface that generally places the germline-encoded CDR1 and CDR2 loops in contact with the conserved helical residues of the MHC, whereas the highly variable, somatically recombined CDR3 loops primarily interact with the peptide. This docking orientation, also called the ''footprint,'' shows a high degree of variability (Ϯ60°) in different pMHC complex...

T Cell–Dendritic Cell Interaction Dynamics during the Induction of Respiratory Tolerance and Immunity

“…However, recent studies clearly demonstrated that additional factors, such as Ag dose, peptide-MHC potency, as well as T cell and DC frequencies, essentially contribute to the stability of early T cell-DC interactions (31)(32)(33)(34). It is therefore not surprising that studies addressing T cell-DC interactions under tolerizing conditions have reported different kinetics of DC-T cell interaction: Hugues et al (11) found a three-phase kinetic of T cell-DC interactions, whereas Zinselmeyer et al (13) describe the existence of an early phase, when T cells form transient contacts at 8 h after Ag delivery, and a late phase characterized by stable contact formation ∼20 h after Ag delivery.…”

Bidirectional binding of invariant chain peptides to an MHC class II molecule

“…Some information could be obtained on MHCII complexes by detecting the bound peptide (17,18). NMR analysis of the MHC protein itself was only possible for the MHCI-β 2 -microglobulin (19-21), a murine single-chain construct of an MHCI heavy chain fragment (22), and a fragment of a murine class II MHC (23). In this study we use backbone NMR assignment of an MHCII subunit to investigate the dynamic reorientation of CLIP variants in complex with HLA-DR1.…”

A Novel MHC-I Surface Targeted for Binding by the MCMV m06 Immunoevasin Revealed by Solution NMR

“…Protein was purified by gel filtration and ion exchange chromatography on mono-Q, showing a single, monodisperse peak in the chromatogram. Isotope-labeled preparations of H2-L d were made with a similar protocol but instead using 13 C-, 15 N-, and 2 H-substituted M9 minimal media to prepare the inclusion bodies as described for m04 (11).…”

“…Toward this end, we prepared samples of the mini-H2-L d construct refolded in vitro with two different peptides. In particular, we used the QL9 peptide (21) (also employed previously to study binding to the 2C TCR (13,16,17)) and a high affinity NIH self-peptide. Although initially, both samples gave well dispersed two-dimensional TROSY spectra, each indicative of a properly conformed peptide⅐ MHC-I complex, the sample prepared with QL9 deteriorated within 1 week at 25°C.…”

“…Previous NMR (11) and x-ray (12) structural studies of the m04 luminal domain concurrently revealed an intricate variant of the Ig-fold that is conserved among members of the m02-m16 immmunoevasin family, including m06, suggesting a conserved mechanism for MHC-I binding. Using high resolution NMR, Varani et al (13) exploited a truncated H2-L d molecule to map the binding site of the 2C T cell receptor on the peptide⅐MHC-I complex. First, they engineered mini-H2-L d , a minimal ␣1␣2 platform molecule derived from the murine H2-L d protein, which lacks the ␣3 domain and does not require the light chain ␤ 2 m for refolding in vitro (14).…”