Sun Yi, Young Michael C, Woodward Claire H, Danon Julia N, Truong Hau, Gupta Sagar, Winters Trenton J, Burslem George, Sgourakis Nikolaos G
bioRxiv. 2023 Mar 18:2023.03.18.533266. doi: 10.1101/2023.03.18.533266.
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 (β microglobulin, β m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/β m 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 β m interacting sites of the peptide binding groove to long-range effects on the α helix and α 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 β-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.
We 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 β m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked β m 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.
I类主要组织相容性复合体（MHC-I）以及负载次优肽、代谢物或糖脂的类MHC分子具有多态性且内在不稳定，这给识别疾病相关抗原和抗原特异性T细胞受体（TCR）带来了根本性挑战，阻碍了自体疗法的发展。在此，我们利用肽与轻链（β2微球蛋白，β2m）亚基之间的正构象变构偶联，通过一个工程化二硫键连接HC/β2m界面上的保守表位，使其与MHC-I重链（HC）结合，从而生成构象稳定的开放型MHC-I分子。生物物理表征表明，与野生型相比，当负载低至中等亲和力的肽时，开放型MHC-I分子是折叠正确且热稳定性增强的蛋白质复合物。利用溶液核磁共振（NMR），我们表征了二硫键对MHC-I结构的构象和动力学的影响，范围从肽结合槽中β2m相互作用位点的局部变化到对α螺旋和α结构域的远程影响。链间二硫键使空的MHC-I分子稳定在一种肽可接受的开放构象中，以促进跨多种人类白细胞抗原（HLA）同种异型的肽交换，涵盖了来自五个HLA-A、六个HLA-B超型以及寡态HLA-Ib分子的代表。我们的结构设计与条件性β肽配体相结合，为生成稳定性增强的随时可负载的MHC-I系统提供了一个通用平台，能够在高度多态的HLA-I同种异型以及寡态非经典分子的背景下，采用一系列方法筛选抗原表位文库并探测多克隆TCR库。
我们概述了一种结构导向的方法，用于生成构象稳定、开放的MHC-I分子，其配体交换动力学增强，涵盖五个HLA-A、所有HLA-B超型以及寡态HLA-Ib同种异型。我们通过溶液核磁共振（NMR）和氢-氘交换质谱（HDX-MS）光谱提供了肽结合与β2m与重链之间正构象变构协同作用的直接证据。我们证明，共价连接的β2m作为一种构象伴侣，通过诱导开放构象并防止内在不稳定的异二聚体不可逆聚集，使空的MHC-I分子稳定在肽可接受状态。我们的研究为MHC-I三元复合物的构象特性提供了结构和生物物理见解，可进一步应用于改进泛HLA等位基因背景下超稳定通用配体交换系统的设计。
