CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Biol Chem. 2011 May 20;286(20):18013-25. doi: 10.1074/jbc.M111.221028. Epub 2011 Mar 30.
The myeloid inhibitory receptor LILRB4 (also called ILT3, LIR-5, CD85k), a member of the leukocyte immunoglobulin-like receptors (LILRs/LIRs), is an important mediator of immune tolerance. Up-regulated on tolerogenic dendritic cells, it has been shown to modulate immune responses via induction of T cell anergy and differentiation of CD8(+) T suppressor cells and may play a role in establishing immune tolerance in cancer. Consequently, characterizing the molecular mechanisms involved in LILRB4 function and in particular its structure and ligands is a key aim but has remained elusive to date. Here we describe the production, crystallization, and structure of the LILRB4 ectodomain to 1.7 Å using an expression strategy involving engineering of an additional disulfide bond in the D2 domain to enhance protein stability. LILRB4 comprises two immunoglobulin domains similar in structure to other LILRs; however, the D2 domain, which is most closely related to the D4 domains of other family members, contains 3(10) helices not previously observed. At the D1-D2 interface, reduced interdomain contacts resulted in an obtuse interdomain angle of ∼107°. Comparison with MHC class I binding Group 1 LILRs suggests LILRB4 is both conformationally and electrostatically unsuited to MHC ligation, consistent with LILRB4 status as a Group 2 LILR likely to bind novel non-MHC class I ligands. Finally, examination of the LILRB4 surface highlighted distinctive surface patches on the D1 domain and D1D2 hinge region, which may be involved in ligand binding. These findings will facilitate our attempts to precisely define the role of LILRB4 in the regulation of immune tolerance.
髓系抑制性受体 LILRB4（也称为 ILT3、LIR-5、CD85k）是白细胞免疫球蛋白样受体（LILRs/LIRs）的成员，是免疫耐受的重要介质。在耐受性树突状细胞上上调，它已被证明通过诱导 T 细胞无能和 CD8+T 抑制细胞的分化来调节免疫反应，并且可能在癌症中发挥建立免疫耐受的作用。因此，表征 LILRB4 功能涉及的分子机制，特别是其结构和配体，是一个关键目标，但迄今为止一直难以实现。在这里，我们描述了使用涉及在 D2 结构域中工程额外二硫键以增强蛋白质稳定性的表达策略，将 LILRB4 胞外结构域生产、结晶和结构解析至 1.7Å。LILRB4 由两个结构类似于其他 LILRs 的免疫球蛋白结构域组成；然而，与其他家族成员的 D4 结构域最密切相关的 D2 结构域包含以前未观察到的 3(10) 螺旋。在 D1-D2 界面处，减少的结构域间接触导致结构域间角度约为 107°。与 MHC 类 I 结合的 Group 1 LILRs 的比较表明，LILRB4 在构象和静电上都不适合 MHC 结合，这与 LILRB4 作为 Group 2 LILR 的状态一致，可能与新型非 MHC 类 I 配体结合。最后，对 LILRB4 表面的检查突出了 D1 结构域和 D1D2 铰链区域的独特表面斑块，这些斑块可能参与配体结合。这些发现将有助于我们尝试精确定义 LILRB4 在调节免疫耐受中的作用。
