Stabilization of a compact conformation of monomeric GroEL at low temperature by adenine nucleotides

AbstractE. coli GroEL chaperonin monomers, isolated after urea-induced dissociation of GroEL14, undergo cold denaturation below 5° C. Above 5°C, these monomers undergo MgATP-dependent self-assembly. We have demonstrated a conformational transition at 0°C induced by interaction of monomeric GroEL with adenine nucleotides. This conformation has a dramatically decreased Stokes radius and enhanced resistance to trypsin but it is slightly less compact than the conformation of monomers at 23°C in the absence of MgATP and it is not capable of spontaneous self-assembly. A second, temperature-dependent conformational change with a transition at about 5°C is required for GroEL to undergo oligomerization

Structural and kinetic basis for heightened immunogenicity of T cell vaccines

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR–peptide–MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)–A2 tumor epitope NY–ESO-1157–165–SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine–tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA–A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials

CD1b-restricted GEM T cell responses are modulated by Mycobacterium tuberculosis mycolic acid meromycolate chains

Tuberculosis, caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in tuberculosis granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents

Crystallization and preliminary X-ray structural studies of a high-affinity CD8αα co-receptor to pMHC

A high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method

Structure and binding kinetics of three different human CD1d-?-galactosylceramide-specific T cell receptors

Invariant human TCR V?24-J?18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-?-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-?-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-?-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3?, CDR3?, and CDR1? interact with ligands presented by CD1d, whereas CDR2? binds to the CD1d ?1 helix. This docking provides an explanation for the dominant usage of V?11 and V?8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-?-GalCer.Abbreviations used: ?-GalCer, ?-galactosylceramide; CDR, complementarity determining region; CNS, Crystallography and NMR system; DN, double negative; ds, disulfide-linked; iNKT, invariant NKT; J, junctional; PC, phosphatidylcholine; rmsd, root mean square deviation; V, variable.<br/