Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
J Biol Chem. 2023 Oct;299(10):105136. doi: 10.1016/j.jbc.2023.105136. Epub 2023 Aug 4.
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 a stable form, independent 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 βm 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 protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.
人 Tapasin（hTapasin）是 MHC-I 分子的主要伴侣分子，使肽加载和抗原库优化跨越 HLA 同种异型。然而，它作为蛋白加载复合物（PLC）的一部分局限于内质网（ER）腔室中，因此在重组形式中表达时高度不稳定。需要额外的稳定辅助因子（如 ERp57）来催化体外肽交换，限制了生成所需抗原特异性的 pMHC-I 分子的用途。在这里，我们表明鸡 Tapasin（chTapasin）可以以稳定的形式以高产量重组表达，而不依赖于共伴侣。chTapasin 可以以低微摩尔范围的亲和力与人类 HLA-B∗37:01 结合形成稳定的三级复合物。通过基于甲基的 NMR 方法进行的生物物理特性分析表明，chTapasin 识别 HLA-B∗37:01 上的保守βm 表位，与先前解决的 hTapasin X 射线结构一致。最后，我们提供了证据表明 B∗37:01/chTapasin 复合物是肽接受的，并且可以在结合高亲和力肽时解离。我们的结果强调了 chTapasin 作为用于旨在扩展人类 MHC-I 和 MHC 样分子上的配体交换功能的蛋白质工程应用的稳定支架的用途。
