Wu Hongyi, Cao Ruiyu, Wei Shukun, Pathan-Chhatbar Salma, Wen Maorong, Wu Bin, Schamel Wolfgang W, Wang Shuqing, OuYang Bo
State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Signalling Research Centers BIOSS and CIBSS, University of Freiburg, Freiburg, Germany; Department of Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany; Centre for Chronic Immunodeficiency (CCI), University Clinics and University of Freiburg, Freiburg, Germany.
J Mol Biol. 2021 Dec 3;433(24):167328. doi: 10.1016/j.jmb.2021.167328. Epub 2021 Oct 22.
T cell receptor (TCR) signaling in response to antigen recognition is essential for the adaptive immune response. Cholesterol keeps TCRs in the resting conformation and mediates TCR clustering by directly binding to the transmembrane domain of the TCRβ subunit (TCRβ-TM), while cholesterol sulfate (CS) displaces cholesterol from TCRβ. However, the atomic interaction of cholesterol or CS with TCRβ remains elusive. Here, we determined the cholesterol and CS binding site of TCRβ-TM in phospholipid bilayers using solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulation. Cholesterol binds to the transmembrane residues within a CARC-like cholesterol recognition motif. Surprisingly, the polar OH group of cholesterol is placed in the hydrophobic center of the lipid bilayer stabilized by its polar interaction with K154 of TCRβ-TM. An aromatic interaction with Y158 and hydrophobic interactions with V160 and L161 stabilize this reverse orientation. CS binds to the same site, explaining how it competes with cholesterol. Site-directed mutagenesis of the CARC-like motif disrupted the cholesterol/CS binding to TCRβ-TM, validating the NMR and MD results.
T细胞受体（TCR）对抗原识别的信号传导对于适应性免疫反应至关重要。胆固醇使TCRs保持静止构象，并通过直接结合TCRβ亚基的跨膜结构域（TCRβ-TM）介导TCR聚集，而硫酸胆固醇（CS）则将胆固醇从TCRβ上置换下来。然而，胆固醇或CS与TCRβ的原子相互作用仍不清楚。在这里，我们使用溶液核磁共振（NMR）光谱和分子动力学（MD）模拟确定了磷脂双层中TCRβ-TM的胆固醇和CS结合位点。胆固醇与类CARC胆固醇识别基序内的跨膜残基结合。令人惊讶的是，胆固醇的极性OH基团位于脂质双层的疏水中心，通过与TCRβ-TM的K154的极性相互作用而稳定。与Y158的芳香相互作用以及与V160和L161的疏水相互作用稳定了这种反向取向。CS与同一位点结合，解释了它如何与胆固醇竞争。类CARC基序的定点诱变破坏了胆固醇/CS与TCRβ-TM的结合，验证了NMR和MD结果。
