Biology subjects

Woodward, C. H.

Publications and source records attributed to Woodward, C. H..

3 recordsLinked to original sources

bioRxiv2024

Regulating IL-2 immune signaling function via a core allosteric structural network

Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2s pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2s therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and excited state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkines core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles. HighlightsO_LINMR and molecular dynamics simulations revealed distinct conformational dynamics and allosteric networks in computationally re-designed IL-2 superkines compared to wild-type IL-2, despite their similar crystal structures. C_LIO_LIThe superkines S1 and S15 exhibit altered sampling of excited state conformations at an intermediate timescale, with slower conformational exchange rates compared to wild-type IL-2. C_LIO_LIA rationally designed mutation (L56A) in the S1 superkines core allosteric network partially reverted its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. C_LIO_LIOur study demonstrates that IL-2 core dynamics play a critical role in receptor binding and signaling function, providing a foundation for engineering more selective IL-2-based immunotherapies. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/617024v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e298adorg.highwire.dtl.DTLVardef@195b602org.highwire.dtl.DTLVardef@1e06205org.highwire.dtl.DTLVardef@4351e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC) and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in stable form, independently of co-chaperones. chTapasin can bind the human HLA-B*37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved {beta}2m epitope on HLA-B*37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B*37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for future protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

immunology↗

bioRxiv2023

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain ({beta}2 microglobulin, {beta}2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/{beta}2m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in {beta}2m interacting sites of the peptide binding groove to long-range effects on the 2-1 helix and 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional {beta}-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance StatementWe outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and {beta}2m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked {beta}2m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.

biochemistry↗