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Structure and mechanism of immunoreceptors: New horizons in T T TT cell and B B BB cell receptor biology and beyond
免疫受体的结构和机制：细胞和细胞受体生物学及其他领域的新视野

Christoph Thomas and Robert Tampé
克里斯托夫-托马斯和罗伯特-坦佩

Abstract 摘要

Immunoreceptors, also named non-catalytic tyrosine-phosphorylated receptors, are a large class of leukocyte cellsurface proteins critically involved in innate and adaptive immune responses. Their most characteristic defining feature is a shared signal transduction machinery where binding events of cell surface-anchored ligands to the small extracellular receptor domains are translated into phosphorylation of conserved tyrosine-containing cytosolic sequence motifs initiating downstream signal transduction cascades. Despite their central importance to immunology, the molecular mechanism of how ligand binding activates the receptors and results in robust intracellular signaling has remained enigmatic. Recent breakthroughs in our understanding of the architecture and triggering mechanism of immunoreceptors come from cryogenic electron microscopy studies of the B B BB cell and T T TT cell antigen receptors.
免疫受体，又称非催化酪氨酸磷酸化受体，是一大类白细胞细胞表面蛋白，主要参与先天性和适应性免疫反应。它们的最大特征是具有共同的信号转导机制，细胞表面锚定配体与小的胞外受体结构域结合后，转化为含有保守的酪氨酸的细胞膜序列基序的磷酸化，从而启动下游信号转导级联。尽管配体对免疫学至关重要，但配体结合如何激活受体并导致强大的细胞内信号转导的分子机制一直是个谜。对细胞和细胞抗原受体的低温电子显微镜研究为我们了解免疫受体的结构和触发机制提供了最新突破。

Addresses 地址

Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Maxvon-Laue Str. 9, 60438 Frankfurt/Main, Germany
德国法兰克福歌德大学生物中心生物化学研究所，Maxvon-Laue Str.
Corresponding author: Tampé, Robert (tampe@em.uni-frankfurt.de) (Tampé R.)
通讯作者：罗伯特-坦佩（) (Tampé R.)
Current Opinion in Structural Biology 2023, 80:102570
结构生物学最新观点 2023，80:102570
This review comes from a themed issue on Membranes (2023)
本评论选自《薄膜》（2023 年）主题专刊
Edited by Simon Newstead and Robert Tampé
编辑：西蒙-纽斯特德和罗伯特-坦佩
For complete overview of the section, please refer the article collection Membranes (2023)
有关该部分的完整概述，请参阅《膜收集》（2023 年）一文。
Available online 20 March 2023
可于 2023 年 3 月 20 日在线查阅
https://doi.org/10.1016/j.sbi. 2023.102570 .2023.102570
0959-440X/© 2023 Elsevier Ltd. All rights reserved.
0959-440X/© 2023 爱思唯尔有限公司。保留所有权利。

Introduction 导言

It was 1897 when Paul Ehrlich, one of the founders of immunology, postulated that cells present “side-chains” on their surface, allowing them to specifically bind toxins, akin to the “lock-and-key” model of enzyme-substrate recognition developed by Hermann Emil Fischer in 1894 [1]. The binding event was suggested by Ehrlich to elicit the production of more “sidechains” and their release into the blood stream to neutralize remaining toxin molecules. By 1900, Ehrlich had renamed the “receptive side-chains” of his theory and thereby coined the term “receptor” still in use today. However, it was not before the 1970s that cellsurface receptors were actually isolated as defined proteins from cell membranes. The existence of the two major antigen receptors in adaptive immunity, the B cell receptor (BCR) and the T cell receptor (TCR), which are the focus of this review, was first demonstrated in 1970 by Raff et al. [2] and Pernis et al. [3] and in 1982 by Allison et al. [4], respectively. We now know that the BCR and TCR are members of a large class of leukocyte cell-surface receptors called immunoreceptors. The almost 100 members of this receptor class are type I or type II membrane proteins that can be divided into different families based on their extracellular domains (ECDs) [5] which are typically built from either immunoglobulin (Ig) folds, C-type lectin domains, or scavenger receptor cysteine-rich domains. Apart from BCR and TGR, prominent immunoreceptors include the Fc receptors, natural killer group 2D (NKG2D), cluster of differentiation 28 (CD28), and programmed cell death protein 1 (PD-1), all fundamentally important in basic and applied biomedicine and life sciences.
1897 年，免疫学奠基人之一保罗-埃利希（Paul Ehrlich）推测，细胞表面存在 "侧链"，可以特异性地与毒素结合，这与赫尔曼-埃米尔-费舍尔（Hermann Emil Fischer）于 1894 年提出的酶-底物识别 "锁-键 "模型相似[1]。埃利希认为，这一结合过程会产生更多的 "侧链"，并将其释放到血液中以中和剩余的毒素分子。到 1900 年，埃利希将其理论中的 "受体侧链 "重新命名，并由此创造了 "受体 "一词，该词一直沿用至今。然而，直到 20 世纪 70 年代，细胞表面受体才真正作为确定的蛋白质从细胞膜中分离出来。1970 年，Raff 等人[2]和 Pernis 等人[3]以及 1982 年 Allison 等人[4]分别首次证明了适应性免疫中两种主要抗原受体--B 细胞受体（BCR）和 T 细胞受体（TCR）--的存在。我们现在知道，BCR 和 TCR 是一大类称为免疫受体的白细胞细胞表面受体的成员。这一类受体的近 100 个成员都是 I 型或 II 型膜蛋白，可根据它们的胞外结构域（ECD）[5] 分成不同的家族，ECD 通常由免疫球蛋白（Ig）褶皱、C 型凝集素结构域或清道夫受体富半胱氨酸结构域构成。除 BCR 和 TGR 外，著名的免疫受体还包括 Fc 受体、天然杀伤群 2D（NKG2D）、分化群 28（CD28）和程序性细胞死亡蛋白 1（PD-1），它们在基础和应用生物医学及生命科学中都具有重要意义。
The different immunoreceptors are subsumed into one class because they share a common set of characteristic features that also distinguishes them from other important receptor classes, i.e., receptor tyrosine kinases, cytokine receptors, integrins, and G proteincoupled receptors (GPCRs): Immunoreceptors lack intrinsic catalytic activity, their ligands are typically plasma membrane-anchored on an interacting cell, and signaling is mediated by phosphorylation of conserved cytosolic, tyrosine-harboring sequence motifs that are either intrinsic to the receptor chain or contained in associated transmembrane signaling subunits [5]. Because of these features, immunoreceptors are also referred to as non-catalytic tyrosine-phosphorylated receptors (NTRs). The cytosolic sequence motifs can be grouped into immunoreceptor tyrosine-based activation motifs (ITAMs), inhibitory motifs (ITIMs), and switch motifs (ITSMs), with ITAMs having the consensus sequence
不同的免疫受体之所以被归为一类，是因为它们具有一系列共同的特征，这些特征也将它们与其他重要的受体类别（即受体酪氨酸激酶、细胞因子受体、整合素和 G 蛋白偶联受体 (GPCR)）区分开来：免疫受体缺乏内在催化活性，它们的配体通常锚定在相互作用细胞的质膜上，信号传递是由保守的细胞膜酪氨酸序列基序磷酸化介导的，这些基序要么是受体链固有的，要么包含在相关的跨膜信号亚基中[5]。由于这些特征，免疫受体也被称为非催化酪氨酸磷酸化受体（NTR）。细胞膜序列基序可分为基于免疫受体酪氨酸的激活基序（ITAM）、抑制基序（ITIM）和开关基序（ITSM）。 ( D / E ) xx X xx ( L ( L ) x 6 − 8 Y xx ( L / I ) ( D / E ) xx X xx ( L ( L ) x 6 − 8 Y xx ( L / I ) (D//E)xx_(X)xx_((L)(L)x_(6-8)Y_(xx)(L//I)(\mathrm{D} / \mathrm{E}) \mathrm{xx}_{\mathrm{X}} \mathrm{xx}_{(\mathrm{L}}(\mathrm{L}) \mathrm{x}_{6-8} \mathbf{Y}_{\mathrm{xx}}(\mathrm{L} / \mathrm{I}) (x being any amino acid). The motif-bearing cytosolic regions are
(x为任意氨基酸）。含图案的细胞膜区域是
intrinsically disordered, and phosphorylation is in most cases catalyzed by discrete lipid-modified, membraneanchored non-receptor tyrosine kinases of the Src family, whereas the antagonistic dephosphorylation is carried out by receptor protein tyrosine phosphatases (RPTPs), such as the leukocyte-specific CD45 and CD148, which are type I transmembrane glycoproteins with very large extracellular regions. The phosphorylated tyrosine residues eventually serve as docking platforms for activating or inhibitory Src homology 2 (SH2) domain-containing downstream signaling proteins. The signaling system can be envisioned as multivalent and highly dynamic, with the activating Src-family kinases and the inhibitory phosphatases being constitutively active [6]. Before ligand binding, phosphatases govern the phosphorylation status of the receptors and suppress signal transduction. According to the “kinetic proofreading” model, the steady-state balance between phosphatases and kinases is shifted towards the latter upon ligand binding and leads to a build-up of phosphorylated ITAMs [5].
在大多数情况下，磷酸化是由离散的脂质修饰、膜锚定的 Src 家族非受体酪氨酸激酶催化的，而拮抗性去磷酸化则是由受体蛋白酪氨酸磷酸酶（RPTPs）进行的，如白细胞特异性 CD45 和 CD148，它们都是 I 型跨膜糖蛋白，具有非常大的胞外区域。磷酸化的酪氨酸残基最终成为激活或抑制含 Src 同源 2（SH2）结构域下游信号蛋白的对接平台。该信号系统可被视为多价和高度动态的，其中激活型 Src 家族激酶和抑制型磷酸酶都具有组成活性 [6]。在配体结合之前，磷酸酶会控制受体的磷酸化状态并抑制信号转导。根据 "动力学校对 "模型，配体结合后，磷酸酶和激酶之间的稳态平衡会向后者倾斜，导致磷酸化的 ITAMs 增加[5]。
While the shared intracellular signaling machinery and downstream signaling pathways of immunoreceptors are well characterized, the mechanism by which ligand binding leads to receptor triggering and results in sustained cytosolic tyrosine phosphorylation has remained enigmatic. Here, we highlight recent advances in our knowledge of immunoreceptor complex assembly and the mechanistic underpinnings of receptor triggering, as revealed by single-particle cryogenic electron microscopy (cryo-EM) studies, particularly on key immunoreceptors, the antigen receptors of B B BB and T lymphocytes.
虽然免疫受体共享的细胞内信号转导机制和下游信号通路已得到充分表征，但配体结合导致受体触发并导致持续胞质酪氨酸磷酸化的机制仍然是个谜。在这里，我们重点介绍了单粒子低温电子显微镜 （cryo-EM） 研究揭示的免疫受体复合物组装和受体触发机制基础的最新进展，特别是关于关键免疫受体，即 B B BB and T lymphocytes.

The assembly principles of the T T TT cell receptor complex
该 T T TT 细胞受体复合物/

The α β T α β T alpha betaT\alpha \beta \mathrm{T} cell receptor (TCR) complex mediates antigen-specific adaptive immune responses against diseased target cells by recognizing specific peptides presented on major histocompatibility complex class I or class II molecules (pMHC I/II) [7,8]. The highly variable, antigen-recognizing subunits of the TCR complex, TCR α α alpha\alpha and TCR β β beta\beta, are disulfide-linked and consist of an extracellular Ig-like variable (V) and constant © domain, a cysteine-containing connecting-peptide linker, followed by a transmembrane ™ helix and a short cytosolic region (Figure 1). The pMHC binding site of TCR α β α β alpha beta\alpha \beta is formed by six complementaritydetermining regions (CDRs), three of which are contributed by each V domain. V α V β α V β alphaVbeta\alpha \mathrm{V} \beta typically docks
这 α β T α β T alpha betaT\alpha \beta \mathrm{T} 细胞受体（TCR）复合物通过识别呈现在主要组织相容性复合物 I 类或 II 类分子（pMHC I/II）上的特异性肽，介导针对病变靶细胞的抗原特异性适应性免疫反应 [7,8]。TCR 复合物中高度可变的抗原识别亚基 TCR α α alpha\alpha 和 TCR β β beta\beta 是二硫键连接的，由细胞外 Ig 样可变（V）。和恒定©结构域、含半胱氨酸的连接肽接头、跨膜™螺旋和短胞质区域组成（图 1）。TCR 的 pMHC 结合位点 α β α β alpha beta\alpha \beta 由六个互补决定区 （CDR）形成，其中三个由每个 V 结构组成。形成，其中三个由每个 V 结构域贡献。V α V β α V β alphaVbeta\alpha \mathrm{V} \beta 通常停靠
Figure 1 图 1
Subunit organization and functional elements of the TCR complex. The α β α β alpha beta\alpha \beta TCR complex is assembled from the two disulfide-bridged (-S-S-) antigen-
TCR复合物的亚基组织和功能元素。 TCR 复合物是由两个二硫键（-S-S-）连接的抗原-TCR 复合物组装而成的。
TCR α α alpha\alpha and TCR β β beta\beta and of CD3 , CD3 δ δ delta\delta, and CD3 γ γ gamma\gamma mediate ligand binding and complex assembly, respectively. The cytosolic regions of the CD3 chains contain conserved sequence motifs called ITAMs (immunoreceptor tyrosine-based activation motifs), which are typical of immunoreceptors. ITAMs mediate receptor signaling by serving, after phosphorylation (yellow dots), as docking platforms for the ζ ζ zeta\zeta-associated protein of 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family member. Active lymphocyte-specific protein tyrosine kinase (Lck) recruited to the receptor complex after ligand engagement by co-receptors can phosphorylate the two tyrosine residues in the ITAMs and the interdomain linker and activation loop of receptor-associated ZAP-70. The Lck-catalyzed phosphorylation of ZAP-70 stabilizes this kinase in its active conformation. Activated ZAP-70 and Lck propagate the signal by instigating various downstream pathways (grey arrows). Phosphorylation by kinase activity is counteracted by receptor protein tyrosine phosphatases. CD3, cluster of differentiation 3.
-70 kDa的相关蛋白（ZAP-70）是脾脏酪氨酸激酶（Syk）家族的成员。淋巴细胞特异性蛋白酪氨酸激酶（Lck）在共受体与配体接合后被招募到受体复合物中，可使受体相关 ZAP-70 的 ITAMs 中的两个酪氨酸残基以及域间连接体和激活环磷酸化。Lck 催化的 ZAP-70 磷酸化可使该激酶稳定在其活性构象中。激活的 ZAP-70 和 Lck 通过启动各种下游通路传播信号（灰色箭头）。激酶的磷酸化活性被受体蛋白酪氨酸磷酸酶抵消。CD3，分化簇 3。
onto pMHC in a diagonal fashion relative to the long axis of the peptide-binding groove
相对于肽结合槽的长轴，以对角线方式连接到 pMHC 上 [ 9 , 10 ] [ 9 , 10 ] [9,10][9,10], and interactions with the peptide are predominantly mediated by the hypervariable CDR3s, while the CDR1s and CDR2s are mainly involved in contacts to the MHC [7]. A salient feature of TCR α β α β alpha beta\alpha \beta complexes is their ability to combine cross-reactivity with high specificity and sensitivity for their agonist pMHC ligands in interactions of medium to low three-dimensional (solution) affinity ( K d ≈ 1 − 10 K d ≈ 1 − 10 K_(d)~~1-10K_{\mathrm{d}} \approx 1-10 μ M μ M muM\mu \mathrm{M} ) with typical maximal half-lives of only several seconds.
以相对于肽结合槽长轴的对角线方式移植到 pMHC 上 [ 9 , 10 ] [ 9 , 10 ] [9,10][9,10] ，与肽的相互作用主要由高变 CDR3 介导，而 CDR1 和 CDR2 主要参与与 MHC 的接触 [7]。TCR 的显著特点 α β α β alpha beta\alpha \beta 复合物是它们在中低三维（溶液）亲和力 （ K d ≈ 1 − 10 K d ≈ 1 − 10 K_(d)~~1-10K_{\mathrm{d}} \approx 1-10 μ M μ M muM\mu \mathrm{M} ），典型的最大半衰期仅为几秒钟。
The full TCR complex is a supramolecular assembly in which TCR α β α β alpha beta\alpha \beta is bound to a combination of four different invariant cluster of differentiation 3 (CD3) chains, which are essential for both TCR α β α β alpha beta\alpha \beta cell-surface expression and signal transduction. The CD3 subunits associated with TCR α β α β alpha beta\alpha \beta form the defined dimeric submodules CD 3 ε γ , CD 3 ε δ CD 3 ε γ , CD 3 ε δ CD3epsi gamma,CD3epsi delta\mathrm{CD} 3 \varepsilon \gamma, \mathrm{CD} 3 \varepsilon \delta, and disulfide-bridged CD3ケケ. Intracellular signal transduction is mediated by the cytosolic regions of the CD3 subunits which contain ITAMs, in contrast to TCR α β α β alpha beta\alpha \beta (Figure 1). The six CD3 signaling chains provide a total of ten ITAMs, three ITAMs in each CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta chain, and one ITAM in CD 3 ε , CD 3 γ CD 3 ε , CD 3 γ CD3epsi,CD3gamma\mathrm{CD} 3 \varepsilon, \mathrm{CD} 3 \gamma, and CD3 3 . The large number of ITAMs contributes to the high sensitivity of the TCR complex. The cytosolic tails of the ITAM-containing CD3 subunits are positively charged and might initially be shielded from phosphorylation by interacting with negatively-charged lipids in the inner leaflet of the plasma membrane [11-13]. Upon binding of agonist pMHC , tyrosine residues in the ITAMs are phosphorylated by the Src-family kinase Lck (lymphocyte-specific protein tyrosine kinase). The co-receptors CD4 and CD 8 , which bind the same pMHC ligand as the receptor but in non-polymorphic regions, augment this process by transiently interacting with Lck. Binding of pMHC thus increases the relative local Lck concentration at the ITAMs of the TCR. Moreover, the co-receptors stabilize the pMHC-TCR complex and thereby increase its lifetime. ITAM phosphorylation leads to recruitment and activation of the auto-inhibited ζ ζ zeta\zeta-associated protein of 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family member [14]. A certain level of basal ITAM phosphorylation and ZAP-70 recruitment is observed even in the absence of ligand binding. Active Lck subsequently phosphorylates regulatory tyrosines in CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta-associated ZAP-70 and thereby fully activates the kinase. Fully active ZAP-70 phosphorylates several different substrates, including the scaffold proteins “linker for the activation of T cells” (LAT), "SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), and the p38 mitogen-activated protein kinase (p38 MAPK) [15]. Phosphorylated LAT and SLP-76 are eventually involved in the activation of different downstream signal transduction enzymes, for example, phospholipase C- γ 1 γ 1 gamma1\gamma 1 (PLC γ 1 γ 1 gamma1\gamma 1 ). Activation of ZAP-70 can also result in insideout signaling in cellular adhesion involving specific integrins when a stronger physical contact between T T TT cell and antigen-presenting cell (APC) and closecontact zones between the membranes of the two cells are established during formation of the so-called immunological synapse or supramolecular activation cluster (SMAC) [16].
完整的 TCR 复合物是一个超分子组装体，其中 TCR α β α β alpha beta\alpha \beta 与四个不同的不变分化簇 3 （CD3）。链的组合结合，这对 TCR 都是必不可少的细胞表面表达和信号转导。与 TCR α β α β alpha beta\alpha \beta 相关联的 CD3 亚基形成了定义的二聚体子模块 CD 3 ε γ , CD 3 ε δ CD 3 ε γ , CD 3 ε δ CD3epsi gamma,CD3epsi delta\mathrm{CD} 3 \varepsilon \gamma, \mathrm{CD} 3 \varepsilon \delta 和二硫键连接的 CD3ケケ 。细胞内信号转导是由 CD3 亚基的胞浆区介导的，其中含有 ITAMs，这与 TCR 不同 α β α β alpha beta\alpha \beta （图 1）。六条 CD3 信号链共提供了十个 ITAM，每条 CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta 链中有三个 ITAM， CD 3 ε , CD 3 γ CD 3 ε , CD 3 γ CD3epsi,CD3gamma\mathrm{CD} 3 \varepsilon, \mathrm{CD} 3 \gamma 和 CD3 3 中有一个 ITAM。大量的 ITAM 使 TCR 复合物具有很高的灵敏度。含 ITAM 的 CD3 亚基的胞浆尾部带正电荷，最初可能通过与质膜内叶中带负电荷的脂质相互作用而免受磷酸化的影响 [11-13]。与激动剂 pMHC 结合后，ITAMs 中的酪氨酸残基会被 Src 家族激酶 Lck（淋巴细胞特异性蛋白酪氨酸激酶）磷酸化。共受体 CD4 和 CD 8 与受体结合了相同的 pMHC 配体，但处于非多态区，它们通过与 Lck 的瞬时相互作用加强了这一过程。因此，pMHC 的结合会增加 TCR ITAMs 上 Lck 的相对局部浓度。此外，共受体还能稳定 pMHC-TCR 复合物，从而延长其寿命。ITAM 磷酸化会导致自身抑制的 ζ ζ zeta\zeta 70 kDa（ZAP-70）相关蛋白（一种脾脏酪氨酸激酶（Syk）家族成员）的招募和激活[14]。即使在没有配体结合的情况下，也能观察到一定程度的基础 ITAM 磷酸化和 ZAP-70 募集。 活性 Lck 随后会使 CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta 相关 ZAP-70 中的调节酪氨酸磷酸化，从而完全激活激酶。完全活化的 ZAP-70 会磷酸化几种不同的底物，包括支架蛋白 "激活 T 细胞的连接蛋白"（LAT）、"含 SH2 结构域的 76 kDa 白细胞蛋白（SLP-76）"和 p38 丝裂原活化蛋白激酶（p38 MAPK）[15]。磷酸化的 LAT 和 SLP-76 最终会参与激活不同的下游信号转导酶，例如磷脂酶 C- γ 1 γ 1 gamma1\gamma 1 （PLC γ 1 γ 1 gamma1\gamma 1 ）。当 T T TT 细胞与抗原递呈细胞（APC）之间的物理接触加强，并且在形成所谓的免疫突触或超分子活化簇（SMAC）的过程中，两个细胞的膜之间建立了紧密接触区时，ZAP-70的活化也会导致细胞粘附中涉及特定整合素的由内而外的信号传导[16]。
While the components of the TCR complex have been known for a long time, insights into the structural principles of their assembly have only recently been gained by a 3.7 Å 3.7 Å 3.7"Å"3.7 \AAÅ cryo-EM reconstruction of an unliganded TCR complex that was stabilized by glutaraldehyde-based crosslinking and whose individual, non-clonotypic subunits were derived from different human cDNA libraries [17]. This reconstruction and the first structure of a signaling-active, tumor-associated pMHC I-specific TCR complex at 3.08 Å [ 18 ] 3.08 Å [ 18 ] 3.08"Å"[18]3.08 \AA[18]Å reveal how TCR α β α β alpha beta\alpha \beta associates with the three dimeric signaling modules CD3 } γ , CD 3 ε δ } γ , CD 3 ε δ }gamma,CD3epsi delta\} \gamma, \mathrm{CD} 3 \varepsilon \delta, and CD3ケY in a 1 : 1 : 1 : 1 1 : 1 : 1 : 1 1:1:1:11: 1: 1: 1 stoichiometry into an octameric assembly (Figure 2a). The structures cover the ECDs with their heterodimer interlocking regions, connecting-peptide regions, and transmembrane domains (TMDs) of all subunits, whereas the cytosolic tails of the CD3 chains are not resolved, due to intrinsic disorder. The structures show that the association between the ECDs is mediated by interactions of the constant domains and connecting-peptide regions of TCR α β α β alpha beta\alpha \beta with the Ig-like domains of CD3 3 δ 3 δ 3delta3 \delta and CD 3 ε γ 3 ε γ 3epsi gamma3 \varepsilon \gamma. In particular, the C domain of TCR α ( C α ) α ( C α ) alpha(Calpha)\alpha(\mathrm{C} \alpha) interacts with the ECD of CD3 3 , while C β C β Cbeta\mathrm{C} \beta interfaces with the ECDs of CD3 3 δ 3 δ 3delta3 \delta and CD3 3 ε 3 ε 3epsi3 \varepsilon. Moreover, the CD 3 ε CD 3 ε CD3epsi\mathrm{CD} 3 \varepsilon ECD of the δ ε δ ε delta epsi\delta \varepsilon heterodimer interfaces with the CD3 γ γ gamma\gamma ECD. The TMDs form an 8 -helix bundle in which the CD3 TM helices are centered around the α β α β alpha beta\alpha \beta chains (Figure 2b). The TM helical interactions are primarily mediated by hydrophobic interactions, but also through salt bridges that are typical of the association between receptor and signaling subunits in immunoreceptors [19].
尽管人们很早就知道TCR复合物的组成成分，但直到最近才通过 3.7 Å 3.7 Å 3.7"Å"3.7 \AAÅ 冷冻电子显微镜重建了未配体的TCR复合物，该复合物是通过戊二醛交联稳定的，其单个非同型亚基来自不同的人类cDNA文库[17]。这一重建结构以及 3.08 Å [ 18 ] 3.08 Å [ 18 ] 3.08"Å"[18]3.08 \AA[18]Å 处的首个信号活性、肿瘤相关 pMHC I 特异性 TCR 复合物结构揭示了 TCR α β α β alpha beta\alpha \beta 如何与三个二聚体信号模块 CD3 } γ , CD 3 ε δ } γ , CD 3 ε δ }gamma,CD3epsi delta\} \gamma, \mathrm{CD} 3 \varepsilon \delta 和 CD3ケY 按 1 : 1 : 1 : 1 1 : 1 : 1 : 1 1:1:1:11: 1: 1: 1 的比例结合成一个八聚体组装体（图 2a）。这些结构涵盖了所有亚基的 ECDs 及其异源二聚体互锁区、连接肽区和跨膜结构域（TMDs），而 CD3 链的细胞膜尾部由于内在无序而未被解析。这些结构表明，ECDs之间的联系是由TCR α β α β alpha beta\alpha \beta 的恒定结构域和连接肽区与CD3 3 δ 3 δ 3delta3 \delta 和CD 3 ε γ 3 ε γ 3epsi gamma3 \varepsilon \gamma 的类Ig结构域的相互作用介导的。特别是，TCR 的 C 结构域 α ( C α ) α ( C α ) alpha(Calpha)\alpha(\mathrm{C} \alpha) 与 CD3 3 的 ECD 相互作用，而 C β C β Cbeta\mathrm{C} \beta 则与 CD3 的 ECD 3 δ 3 δ 3delta3 \delta 和 CD3 的 ECD 3 ε 3 ε 3epsi3 \varepsilon 相互作用。此外， δ ε δ ε delta epsi\delta \varepsilon 杂二聚体的 CD 3 ε CD 3 ε CD3epsi\mathrm{CD} 3 \varepsilon ECD 与 CD3 γ γ gamma\gamma ECD 相互连接。TMD 形成一个 8 螺旋束，其中 CD3 TM 螺旋以 α β α β alpha beta\alpha \beta 链为中心（图 2b）。 TM 螺旋相互作用主要由疏水相互作用介导，但也通过免疫受体中受体和信号亚基之间的典型关联盐桥进行[19]。
While the structure of an unliganded TCR complex significantly extended our understanding of immunoreceptor complex assembly, the pivotal question of how ligand engagement activates the receptor complex remained open. In a follow-up study, cryo-EM reconstructions of the unliganded TCR complex were described at significantly higher resolutions of 3.2 and 3.1 Å 3.1 Å 3.1"Å"3.1 \AAÅ, respectively [20], uncovering two cholesterol molecules bound between the TM helices of TCR α β α β alpha beta\alpha \beta, CD 3 γ CD 3 γ CD3gamma\mathrm{CD} 3 \gamma, and CD3 3 (Figure 2c and d). Surprisingly, when the authors mutated two key TCR β β beta\beta residues located mainly in the first cholesterol binding site, the second cholesterol molecule was no longer visible in the corresponding cryo-EM reconstruction and was presumably replaced by a phospholipid. Most notably, the C-terminal portion of the cholesterol-interacting TM helix of CD3 ζ ζ zeta\boldsymbol{\zeta} was shifted in the mutant structure, causing small
虽然未加载配体的 TCR 复合物结构极大地扩展了我们对免疫受体复合物组装的理解，但配体接合如何激活受体复合物这一关键问题仍然悬而未决。在一项后续研究中，对未加载配体的 TCR 复合物进行了低温电子显微镜重构，分辨率大大提高，达到了 3.2和 3.1 Å 3.1 Å 3.1"Å"3.1 \AAÅ [20]，发现了结合在TCR α β α β alpha beta\alpha \beta , CD 3 γ CD 3 γ CD3gamma\mathrm{CD} 3 \gamma 和CD3 3的TM螺旋之间的两个胆固醇分子（图2c和d）。令人惊讶的是，当作者突变了主要位于第一个胆固醇结合位点的两个关键 TCR β β beta\beta 残基后，在相应的冷冻电镜重建中就看不到第二个胆固醇分子了，估计是被磷脂取代了。最值得注意的是，在突变体结构中，CD3 ζ ζ zeta\boldsymbol{\zeta} 与胆固醇相互作用的 TM 螺旋的 C 端部分发生了偏移，导致小分子胆固醇的结合位点发生了改变。
Figure 2 图 2
Single-particle cryo-EM reconstructions of the unliganded TCR complex. (a) The 3.7 Å 3.7 Å 3.7"Å"3.7 \AAÅ reconstruction of the unliganded TCR complex viewed parallel to the membrane plane. (b) Cytosolic view of the TM helices in the TCR complex structure shown in (a). © Cryo-EM structure of the unliganded TCR complex with two associated cholesterol molecules, shown with their van-der-Waals surface (yellow). Overall resolution and PDB accession code of the structures shown in (a) and © are shown in parentheses. (d) Zoom-in of the cholesterol-binding sites between the TM helices of CD3 γ γ gamma\gamma and one of the CD3そ subunits (CD3そ’). The two cholesterol molecules (yellow) and side chains of cholesterol-interacting protein residues are shown as sticks. Structural images were created using ChimeraX [59].
未加载配体的 TCR 复合物的单颗粒冷冻电镜重构。(a) 平行于膜面观察未连接 TCR 复合物的 3.7 Å 3.7 Å 3.7"Å"3.7 \AAÅ 重建图。(b) (a) 中所示 TCR 复合物结构中 TM 螺旋的细胞膜视图。©带有两个相关胆固醇分子的未连接 TCR 复合物的冷冻电镜结构，显示的是其范德华表面（黄色）。括号中显示的是 (a) 和 © 中所示结构的整体分辨率和 PDB 加入代码。(d）CD3 γ γ gamma\gamma 的 TM 螺旋与 CD3 そ 亚基之一（CD3そ'）之间的胆固醇结合位点放大图。两个胆固醇分子（黄色）和与胆固醇有相互作用的蛋白质残基侧链以棒状显示。结构图像使用 ChimeraX [59] 绘制。
rearrangements in the TM helices of TCR α α alpha\alpha and the second CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta chain. Based on these findings, in combination with results from biochemical experiments analyzing the function of binding-site mutants, the authors conclude that cholesterol is a key mediator of TCR signaling, arresting the TCR complex in an inactive conformation. pMHC binding is proposed to activate TCR signaling by inducing subtle structural changes in the ECDs that trigger cholesterol release and rearrangements in the TM helices, eventually rendering the membrane-associated, ITAM-containing cytosolic tails of the CD3 subunits accessible for phosphorylation by Lck. Although there is previous experimental support for the notion that cholesterol negatively regulates the TCR complex [21,22], the exact role of this sterol lipid in pMHC-triggered TCR activation remains to be further scrutinized.
TCR α α alpha\alpha 的TM螺旋和第二条 CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta 链的重排。根据这些发现，结合分析结合位点突变体功能的生化实验结果，作者得出结论：胆固醇是 TCR 信号转导的关键介质，它能使 TCR 复合物处于非活性构象。pMHC 的结合被认为是通过诱导 ECDs 发生微妙的结构变化来激活 TCR 信号转导，这种变化会触发胆固醇释放和 TM 螺旋的重排，最终使 CD3 亚基的膜结合、含 ITAM 的胞浆尾部可被 Lck 磷酸化。虽然胆固醇对 TCR 复合物有负向调节作用的观点已得到实验支持 [21,22]，但这种甾醇脂质在 pMHC 触发的 TCR 激活中的确切作用仍有待进一步研究。

The architecture of the B B BB cell receptor complex
B B BB 细胞受体复合体的结构

The B cell receptor (BCR) complex mediates antigentriggered proliferation and differentiation of B lymphocytes into antibody-secreting plasma cells and is thus a key constituent of the adaptive immune system. This supramolecular assembly is composed of the antigenrecognizing membrane-bound immunoglobulin (mIg) and a disulfide-linked heterodimer of the ITAM-bearing transmembrane signaling chains Ig α Ig α Ig alpha\operatorname{Ig} \alpha and Ig β Ig β Ig beta\operatorname{Ig} \beta [23]. Although basic structural features of the BCR complex
B 细胞受体（BCR）复合物介导抗原诱导的 B 淋巴细胞增殖和分化为分泌抗体的浆细胞，因此是适应性免疫系统的关键组成部分。这种超分子组合由抗原识别膜结合免疫球蛋白（mIg）和ITAM跨膜信号链 Ig α Ig α Ig alpha\operatorname{Ig} \alpha 和 Ig β Ig β Ig beta\operatorname{Ig} \beta 的二硫键异二聚体组成[23]。尽管 BCR 复合物的基本结构特征
are known, the architecture of the fully assembled mIg Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta complex has remained elusive. Three current studies describe single-particle cryo-EM reconstructions of the IgM- and IgG-BCR complexes at resolutions between 3.0 and 3.6 Å 3.6 Å 3.6"Å"3.6 \AAÅ [24-26] (Figure 3). The reconstructions unveil that the mIg and Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta assemble through interactions between their ECDs, membraneproximal connecting-peptide regions, and the TM helices. The fragment crystallizable ( Fc ) region of mIg stabilizes the complex via contacts with the ECDs of Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta, while the four TM helices form a tight bundle facilitated by conserved hydrophobic and polar interactions, rationalizing the crucial role of the TM region for complex assembly [23]. Interestingly, in the juxtamembrane linker region, one of the mIg chains is threaded in between the two signaling subunits, indicating that the heavy chain of mIg and Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta folds simultaneously during assembly. Just like for the TCR complex, the intracellular domains (ICDs) of the receptor complex subunits are not resolved due to their intrinsic flexibility, with the notable exception of the Ig β ICD Ig β ICD Ig betaICD\operatorname{Ig} \beta \mathrm{ICD} in one of the structures [26]. The ICD is visible as weak map region next to the Ig β Ig β Ig beta\operatorname{Ig} \beta TM helix and can be modeled as the corresponding ITAM motif adopting a possibly autoinhibitory α α alpha\alpha-helical hairpin conformation [26]. This autoinhibitory conformation would suggest a structural change in the ICDs upon antigen binding. Another remarkable finding gained from the BCR complex structures is that the ECDs of IgM- and IgG-BCR differ in their location and orientation relative to Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta : The IgM-ECDs are positioned close to the membrane and lateral with respect to Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta, whereas the IgG IgG IgG\operatorname{IgG}-ECDs are rest on top of Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta. It will be interesting to investigate if this structural divergence can be connected to functional differences of the two receptor isotypes. But above all, future structural studies of the liganded BCR complex will have to address the question of how antigen recognition at the flexibly linked fragment antigen-binding (Fab) regions is translated into cytosolic signaling.
但是，完全组装的 mIg Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 复合物的结构仍然难以捉摸。目前有三项研究描述了分辨率介于 3.0 和 3.6 Å 3.6 Å 3.6"Å"3.6 \AAÅ 之间的 IgM- 和 IgG-BCR 复合物的单颗粒冷冻电镜重构 [24-26]（图 3）。重建结果表明，mIg 和 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 是通过其 ECD、膜近端连接肽区和 TM 螺旋之间的相互作用组装在一起的。mIg 的可结晶片段（Fc）区域通过与 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 的 ECD 接触来稳定复合物，而四个 TM 螺旋则通过保守的疏水和极性相互作用形成紧密的束，从而合理解释了 TM 区域在复合物组装中的关键作用 [23]。有趣的是，在并膜连接区，一条 mIg 链被穿在两个信号亚基之间，这表明 mIg 和 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 的重链在组装过程中同时折叠。与 TCR 复合物一样，受体复合物亚基的胞内结构域（ICD）也因其内在的灵活性而无法解析，但其中一个结构中的 Ig β ICD Ig β ICD Ig betaICD\operatorname{Ig} \beta \mathrm{ICD} 明显例外[26]。ICD在 Ig β Ig β Ig beta\operatorname{Ig} \beta TM螺旋旁边的弱图区可见，可被模拟为相应的ITAM图案，采用可能具有自动抑制作用的 α α alpha\alpha 螺旋发夹构象[26]。这种自动抑制构象表明，ICDs 在与抗原结合后会发生结构变化。 从 BCR 复合物结构中获得的另一个重要发现是，IgM-BCR 和 IgG-BCR 的 ECD 相对于 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 的位置和方向不同：IgM-ECDs 的位置靠近膜，相对于 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 是横向的，而 IgG IgG IgG\operatorname{IgG} -ECDs则位于 Ig α − Ig β Ig α − Ig β Ig alpha-Ig beta\operatorname{Ig} \alpha-\operatorname{Ig} \beta 的顶部。研究这种结构差异是否与两种受体异型的功能差异有关，将是一件有趣的事情。但最重要的是，未来对配体 BCR 复合物的结构研究必须解决这样一个问题，即在灵活连接的片段抗原结合（Fab）区域的抗原识别是如何转化为细胞膜信号的。

How does ligand binding trigger immunoreceptor signaling?
配体结合如何触发免疫受体信号？

Membrane receptors often undergo ligand-induced conformational changes and thereby propagate the signal of the binding event into the cell to actuate downstream signaling cascades [27]. In other cases, receptor dimerization upon ligand binding initiates intracellular signaling events [28]. However, despite extensive research, it is still unclear how ligand binding triggers immunoreceptor signaling. Several models of immunoreceptor triggering have been proposed with respect to the TCR complex: One early concept focused on enhanced recruitment of the ITAM-phosphorylating kinase Lck via the co-receptors CD4 and CD8 [29]. Another more recent model suggests that TCR signaling is triggered by clustering of TCR complexes [30,31], in which clustering or induced proximity of receptors would somehow result in increased phosphorylation of ITAMs. This model is challenged by the low copy number of agonistic pMHC complexes on target cells and by the observation that in CD + + T CD + + T CD^(+)^(+)T\mathrm{CD}^{+}{ }^{+} \mathrm{T} cells, only three
膜受体通常会发生配体诱导的构象变化，从而将结合事件的信号传入细胞，激活下游信号级联[27]。在其他情况下，配体结合后受体二聚化会启动胞内信号传导事件 [28]。然而，尽管进行了广泛的研究，配体结合如何触发免疫受体信号仍不清楚。针对 TCR 复合物提出了几种免疫受体触发模型：早期的一个概念侧重于通过共受体 CD4 和 CD8 增强 ITAM 磷酸化激酶 Lck 的招募 [29]。另一种较新的模型认为，TCR 信号转导是由 TCR 复合物的集群触发的[30,31]，其中受体的集群或诱导接近将以某种方式导致 ITAM 磷酸化的增加。这一模型受到了靶细胞上激动型 pMHC 复合物低拷贝数的挑战，而且在 CD + + T CD + + T CD^(+)^(+)T\mathrm{CD}^{+}{ }^{+} \mathrm{T} 细胞中，只有三个受体被磷酸化[30,31]。
Figure 3 图 3
Single-particle cryo-EM reconstructions of the unliganded IgM IgM IgM\operatorname{IgM} - and lg ⁡ G − B C R lg ⁡ G − B C R lg G-BCR\lg G-B C R complexes. The lg ⁡ M lg ⁡ M lg M\lg M-BCR complex in (a) and IgG-BCR complex in (b) are viewed parallel to the membrane plane. The antigen-binding subunits are shown in red and orange, respectively, while the signaling subunits lg ⁡ α lg ⁡ α lg alpha\lg \alpha and lg ⁡ β lg ⁡ β lg beta\lg \beta are depicted in different shades of blue. Overall resolution and PDB accession code of the structures are shown in parentheses. Structural images were prepared using ChimeraX [59].
未连接的 IgM IgM IgM\operatorname{IgM} - 和 lg ⁡ G − B C R lg ⁡ G − B C R lg G-BCR\lg G-B C R 复合物的单颗粒冷冻电镜重构。(a) 中的 lg ⁡ M lg ⁡ M lg M\lg M -BCR 复合物和 (b) 中的 IgG-BCR 复合物与膜平面平行。抗原结合亚基分别以红色和橙色显示，而信号亚基 lg ⁡ α lg ⁡ α lg alpha\lg \alpha 和 lg ⁡ β lg ⁡ β lg beta\lg \beta 则以不同深浅的蓝色显示。括号内显示的是结构的整体分辨率和 PDB 加入代码。结构图像使用 ChimeraX [59] 绘制。
pMHC ligands can be sufficient for target cell killing without formation of the immunological synapse [32]. And the latest experimental evidence coming from single-molecule brightness and coincidence analysis and fluorescence energy transfer (FRET) studies on living T cells suggests that within the active immunological synapse, monomeric TCR complexes are responsible for recognizing antigenic pMHCs [33]. In a third concept of TCR triggering, the TCR has been proposed to function similarly to other receptor systems, through allosteric
pMHC 配体足以在不形成免疫突触的情况下杀死靶细胞[32]。对活体 T 细胞进行的单分子亮度和巧合分析以及荧光能量转移（FRET）研究提供的最新实验证据表明，在活跃的免疫突触中，单体 TCR 复合物负责识别抗原 pMHC [33]。在 TCR 触发的第三个概念中，有人提出 TCR 的功能与其他受体系统类似，通过异位
Figure 4 图 4
Cryo-EM structure of the fully assembled pMHC-bound TCR complex. (a) The individual protein chains of the receptor and MHC I ligand are shown in ribbon representation and viewed parallel to the plasma membrane plane. The antigenic peptide is depicted with its van-der-Waals surface (yellow). Overall resolution and PDB accession code of the structure are shown in parentheses. (b) Structural differences between the pMHC-bound TCR complex (PDB code: 7PHR) and the unliganded TCR complex (PDB code: 6JXR) mapped onto the liganded complex. Thickness and color of the putty cartoon correspond to the distance between corresponding C α C α Calpha\mathrm{C} \alpha atoms after overall superposition of the two structures (see reference helix at bottom). The pMHC ligand is shown as green/yellow ribbon. © Structural differences between the pMHC-bound TCR complex (PBD code: 7PHR) and the unliganded, cholesterol-bound structure (PDB code: 7FJD) mapped onto the liganded complex. Thickness and color of the putty cartoon correspond to the distance between corresponding C α C α Calpha\mathrm{C} \alpha atoms after superposition of the TM helices in the two structures (see reference helix at bottom). The pMHC ligand is shown as green/yellow ribbon. (d) Alternative models of TCR activation propose that allosteric changes in the TCR complex upon pMHC I engagement make the ITAM-containing cytosolic regions accessible for phosphorylation and downstream signal transduction. The ITAMs in the cytosolic regions of the CD3 subunits are indicated by red rectangles. (e) According to the “kinetic-segregation” model of TCR activation, the receptor protein tyrosine phosphatase CD45 is excluded from the close contact zone formed between antigen-presenting cell (APC) and T cell, due to its large ectodomain (note that only ectodomains d1-d4 are schematically shown). This causes a local shift in the balance between phosphatases and kinases and leads to stable ITAM phosphorylation and downstream signaling, if the pMHC-TCR complex and the close contact zone have sufficiently long lifetimes. The ITAMs in the cytosolic regions of the CD3 subunits are indicated by red rectangles. P1/P2, phosphatase domains of CD45. Structural images of panel (a) were created using ChimeraX [59]. Panels (b) and © were prepared using PyMOL.
完全组装的 pMHC 结合 TCR 复合物的冷冻电镜结构。(a) 受体和 MHC I 配体的单个蛋白链以带状表示，平行于质膜平面观察。抗原肽以范德华表面（黄色）表示。括号内为该结构的整体分辨率和 PDB 加入代码。(b) pMHC 结合的 TCR 复合物（PDB 代码：7PHR）与未配体的 TCR 复合物（PDB 代码：6JXR）之间的结构差异映射到配体复合物上。油灰漫画的厚度和颜色对应于两个结构整体叠加后相应 C α C α Calpha\mathrm{C} \alpha 原子之间的距离（见底部的参考螺旋）。pMHC 配体显示为绿色/黄色色带。pMHC 结合的 TCR 复合物（PBD 代码：7PHR）与未配体、胆固醇结合的结构（PDB 代码：7FJD）映射到配体复合物上的结构差异。油灰漫画的厚度和颜色对应于两个结构中 TM 螺旋叠加后相应 C α C α Calpha\mathrm{C} \alpha 原子间的距离（见底部的参考螺旋）。pMHC 配体显示为绿色/黄色色带。(d) 另一种 TCR 激活模型认为，当 pMHC I 与 TCR 复合物接合时，TCR 复合物中的异构变化会使含 ITAM 的胞浆区域发生磷酸化和下游信号转导。CD3 亚基胞浆区的 ITAM 用红色矩形表示。 (e) 根据 TCR 激活的 "动力学-分离 "模型，受体蛋白酪氨酸磷酸酶 CD45 因其大的外显子域（注意图中仅显示了外显子域 d1-d4）而被排除在抗原提呈细胞（APC）和 T 细胞之间形成的密切接触区之外。如果 pMHC-TCR 复合物和密切接触区的寿命足够长，这将导致磷酸酶和激酶之间的平衡发生局部变化，并导致稳定的 ITAM 磷酸化和下游信号传导。红色矩形表示 CD3 亚基胞浆区域的 ITAM。P1/P2，CD45 的磷酸酶结构域。面板（a）的结构图像由 ChimeraX [59] 绘制。(b)和©板块使用 PyMOL 绘制。
changes in TCR α β α β alpha beta\alpha \beta upon agonistic ligand engagement that are transmitted to the CD3 subunits [34,35]. The structural changes in the CD3 subunits would then facilitate Lck-catalyzed phosphorylation of the cytosolic ITAMs. Crystallographic studies, NMR experiments, and fluorescence measurements suggested pMHC binding-induced conformational changes for the TCR β β beta\beta FG loop [36], TCR α α alpha\alpha AB loop [ 36 , 37 ] [ 36 , 37 ] [36,37][36,37], and the TCR β α A β α A beta alpha A\beta \alpha A helix [ 36 , 38 ] [ 36 , 38 ] [36,38][36,38] in soluble TCR α β α β alpha beta\alpha \beta. Yet, these studies did not demonstrate that the described conformational changes are actually communicated to the CD3 subunits to instigate signaling. A fourth concept of TCR activation, the so-called kinetic-segregation model [39], postulates that upon pMHC recognition, RPTPs are sterically excluded from the close-contact zones due to the size of their ECDs, which are much larger than those of immunoreceptors [39]. The large ECDs of the tyrosine phosphatases are not compatible with the 14 − 16 nm 14 − 16 nm 14-16nm14-16 \mathrm{~nm} gap between the two cell membranes anchoring the immunoreceptor-ligand complex. This size-based exclusion of phosphatases from the closecontact zones, which could still be accessed by Lck, would allow the T cell to get activated by recruitment and phosphorylation of ZAP-70 and downstream adapter proteins [40]. In essence, the segregation would lead to a shift in the metastable balance between phosphatase and kinase activity towards ITAM phosphorylation. Experimental evidence for this model has come from different studies demonstrating that the tyrosine phosphatases CD45 and CD148 segregate from the TCR upon ligand engagement [41-46]. Furthermore, elongated forms of the pMHC ligand inhibit TCR signaling [47-49]. In agreement with the model, the potency of chimeric antigen receptor (CAR) T cells is also influenced by the epitope distance to the APC membrane [50,51]. Interestingly, a similar signaling behavior has been observed for other immunoreceptors of the CD28 and the C-type lectin domain classes [52-54], indicating that the kinetic-segregation model not only applies to TCR signaling, but might be valid for immunoreceptors in general [5]. The segregation process is most likely accompanied by membrane bending leading to mechanical forces acting upon the receptor-ligand complex. TCRs have been proposed to form so-called catch bonds [ 55 , 56 ] [ 55 , 56 ] [55,56][55,56], which are interactions stabilized by these mechanical forces.
当激动配体参与时，TCR α β α β alpha beta\alpha \beta 的变化会传递到 CD3 亚基 [34,35]。CD3 亚基的结构变化会促进 Lck 催化的细胞膜 ITAMs 磷酸化。晶体学研究、核磁共振实验和荧光测量结果表明，pMHC 结合会诱导 TCR β β beta\beta FG 环 [36]、TCR α α alpha\alpha AB 环 [ 36 , 37 ] [ 36 , 37 ] [36,37][36,37] 和可溶性 TCR α β α β alpha beta\alpha \beta 中的 TCR β α A β α A beta alpha A\beta \alpha A 螺旋 [ 36 , 38 ] [ 36 , 38 ] [36,38][36,38] 发生构象变化。然而，这些研究并没有证明所述构象变化实际上传递给了 CD3 亚基，从而启动了信号传导。TCR 激活的第四个概念，即所谓的动力学分离模型[39]，假定在 pMHC 识别后，RPTPs 由于其 ECDs 的大小（远大于免疫受体的 ECDs）而被立体地排除在密切接触区之外[39]。酪氨酸磷酸酶的大 ECD 与锚定免疫受体-配体复合物的两层细胞膜之间的 14 − 16 nm 14 − 16 nm 14-16nm14-16 \mathrm{~nm} 间隙不相容。这种以尺寸为基础将磷酸酶排除在紧密接触区（Lck 仍可进入该区）的做法，可使 T 细胞通过 ZAP-70 和下游适配蛋白的招募和磷酸化而被激活 [40]。从本质上讲，分离将导致磷酸酶和激酶活性之间的可变平衡向 ITAM 磷酸化转变。这一模型的实验证据来自不同的研究，这些研究表明，酪氨酸磷酸酶 CD45 和 CD148 在配体接合时会从 TCR 中分离出来 [41-46]。此外，pMHC 配体的拉长形式会抑制 TCR 信号传导 [47-49]。 与该模型一致的是，嵌合抗原受体（CAR）T 细胞的效力也受表位与 APC 膜距离的影响 [50,51]。有趣的是，在 CD28 和 C 型凝集素结构域类的其他免疫受体中也观察到了类似的信号传导行为 [52-54]，这表明动力学分离模型不仅适用于 TCR 信号传导，也可能适用于一般的免疫受体 [5]。分离过程很可能伴随着膜弯曲，导致机械力作用于受体配体复合物。有人提出 TCR 可形成所谓的 "捕捉键" [ 55 , 56 ] [ 55 , 56 ] [55,56][55,56] ，即通过这些机械力稳定的相互作用。
A recent cryo-EM structure at 3.08 Å 3.08 Å 3.08"Å"3.08 \AAÅ resolution of a signaling-active, fully assembled TCR complex bound to a human class I pMHC helps to narrow down the possible mechanisms of T cell activation [18] (Figure 4a). To obtain a stable pMHC-TCR complex for cryo-EM analysis, an affinity-matured, tumor-reactive TGR was employed. The structure allows a detailed analysis of the connecting-peptide linker regions stabi-
最近，一个信号活跃、完全组装的 TCR 复合物与人类 I 类 pMHC 结合的低温电子显微镜（cryo-EM） 3.08 Å 3.08 Å 3.08"Å"3.08 \AAÅ 分辨率结构有助于缩小 T 细胞活化的可能机制[18]（图 4a）。为了获得稳定的 pMHC-TCR 复合物以进行冷冻电镜分析，我们使用了亲和性成熟的肿瘤反应 TGR。通过该结构可以详细分析连接肽链区，从而稳定 pMHC-TCR 复合物。
modules for TCR complex assembly. Notably, cholesterol is bound in the same region between the TM helices of TCR α β , CD 3 γ α β , CD 3 γ alpha beta,CD3gamma\alpha \beta, \mathrm{CD} 3 \gamma, and CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta as noticed for the unliganded complex [20]. Strikingly, the pMHC-TCR complex exhibits a tilt angle of ∼ 60 ∘ ∼ 60 ∘ ∼60^(@)\sim 60^{\circ} relative to the membrane. This tilt angle might enable the TCR to efficiently scan tens of thousands of pMHC complexes to identify the cognate 2 − 10 2 − 10 2-102-10 antigenic complexes on the target cell, similar to finding the needle in a haystack. However, this would imply that optimal TCR scanning is only achieved when the membranes of T cell and target cell are pre-aligned. Perhaps the most striking revelation offered by the pMHC-ligated reconstruction of the TCR complex is that upon superposition with the unliganded complexes [17,20], only minor structural differences are observed in the TM helices (Figure 4b,c). Surprisingly, the recent cholesterol-bound structures of the unliganded complex [20] (Figure 4c) differ in their ectodomains from the original ligand-free structure [17] (Figure 4b). We have currently no explanation for this unexpected discrepancy in the same, unliganded TCR complex. Yet, importantly, the TMD region still remains virtually unchanged. This finding strongly suggests that activation of the TCR complex upon pMHC binding is triggered not by allosteric changes in the TMD region (Figure 4d), in contrast to other classes of signaling receptors, but according to the “kinetic segregation” model (Figure 4e). It also means that cholesterol could have more of a general structural role in the complex, but it may also influence the assembly of the receptor complex, rather than locking the TCR complex in an inactive conformation, as it has been proposed based on mutagenesis data [20]. The possibility that affinity maturation of the TCR utilized in this work somehow prevented conformational changes upon pMHC binding could be ruled out by molecular-dynamics simulations in which the TCR was mutated back to the wildtype receptor in silico and shown to behave like the affinitymatured version. In addition, the absence of any significant antigen binding-induced structural changes in the components of the TCR complex have recently been independently confirmed by cryo-EM structures of two other distinct liganded TCR complexes exhibiting more typical TCR affinities [57]. In summary, the observation that TCR complexes can function as signal transducers without ligand-induced structural changes indicates that TCR triggering is most likely happening according to the kinetic-segregation model.
TCR 复合物组装的模块。值得注意的是，胆固醇结合在 TCR α β , CD 3 γ α β , CD 3 γ alpha beta,CD3gamma\alpha \beta, \mathrm{CD} 3 \gamma 和 CD 3 ζ CD 3 ζ CD3zeta\mathrm{CD} 3 \zeta 的 TM 螺旋之间的相同区域，这与未加载载体的复合物[20]所注意到的相同。引人注目的是，pMHC-TCR 复合物相对于膜的倾斜角度为 ∼ 60 ∘ ∼ 60 ∘ ∼60^(@)\sim 60^{\circ} 。这种倾斜角可能使 TCR 能够有效地扫描数以万计的 pMHC 复合物，以识别靶细胞上的同源 2 − 10 2 − 10 2-102-10 抗原复合物，类似于大海捞针。然而，这意味着只有当 T 细胞膜和靶细胞膜预先对准时，才能实现最佳的 TCR 扫描。也许 pMHC 连接的 TCR 复合物重构提供的最惊人的启示是，在与未连接的复合物叠加后[17,20]，只观察到 TM 螺旋的微小结构差异（图 4b,c）。令人惊讶的是，未配体复合物的最新胆固醇结合结构[20]（图 4c）与最初的无配体结构[17]（图 4b）在外延上有所不同。我们目前还无法解释在相同的无配体 TCR 复合物中出现这种意外差异的原因。但重要的是，TMD 区域几乎没有变化。这一发现有力地表明，与其他类别的信号受体不同，TCR 复合物与 pMHC 结合后的激活不是由 TMD 区域的异生变化（图 4d）触发的，而是根据 "动力学分离 "模型（图 4e）触发的。这也意味着胆固醇可能在复合物中更多地发挥一般结构作用，但它也可能影响受体复合物的组装，而不是像根据诱变数据提出的那样，将 TCR 复合物锁定在非活性构象中[20]。 分子动力学模拟将 TCR 突变回野生型受体，并显示其行为与亲和成熟型类似，从而排除了这项研究中使用的 TCR 亲和成熟在某种程度上阻止了 pMHC 结合时构象变化的可能性。此外，TCR 复合物各组分中没有任何由抗原结合引起的明显结构变化，这一点最近已被另外两种不同配体的 TCR 复合物的低温电子显微镜结构所证实，这两种复合物表现出更典型的 TCR 亲和性[57]。总之，观察到 TCR 复合物可以在没有配体诱导的结构变化的情况下发挥信号转导功能，这表明 TCR 触发很可能是根据动力学-分离模型进行的。

Conclusion 结论

As key components of the adaptive immune system, the B - and T cell receptors are arguably two of the most important receptors in human physiology. CryoEM , as in so many other areas of structural biology, has opened new avenues in studying these receptor systems, delivering seminal insights that go beyond their ECDs and appreciate the intricate interaction network of these supramolecular transmembrane assemblies. As far as their ligand-mediated activation is concerned, the recent pMHC-bound TCR complex
作为适应性免疫系统的关键组成部分，B 细胞和 T 细胞受体可以说是人类生理学中最重要的两种受体。低温电子显微镜与结构生物学的许多其他领域一样，为研究这些受体系统开辟了新的途径，提供了超越其 ECD 的开创性见解，使人们了解到这些超分子跨膜组装体错综复杂的相互作用网络。就其配体介导的激活而言，最近的 pMHC 结合 TCR 复合物
structures indicate that it is instigated without significant conformational changes relevant to signal transduction. A more advanced understanding of the BCR complex might facilitate vaccine development and help to fight autoimmune diseases and BCRdriven malignancies, while thorough analyses of the mechanisms underlying TCR activation are also expected to have direct clinical implications, as illustrated by chimeric antigen receptor T (CAR-T) cells [58] and catch bond-engineered TCRs [56]. The structure of the ligand-bound receptor complexes might serve as a springboard to further explore ligandmediated receptor triggering, including the intrinsically disordered cytosolic regions, and harness the full therapeutic potential of immunoreceptors.
结构表明，它是在与信号转导相关的构象没有发生重大变化的情况下启动的。正如嵌合抗原受体 T（CAR-T）细胞[58]和捕获键工程 TCR [56]所示，对 BCR 复合物更深入的了解可能会促进疫苗的开发，并有助于对抗自身免疫性疾病和 BCR 驱动的恶性肿瘤。配体结合受体复合物的结构可作为跳板，进一步探索配体介导的受体触发，包括内在紊乱的胞浆区，并充分发挥免疫受体的治疗潜力。

Editorial disclosure statement
编辑披露声明

Given his role as Guest Editor, Robert Tampé had no involvement in the peer review of the article and has no access to information regarding its peer-review. Full responsibility for the editorial process of this article was delegated to Simon Newstead.
鉴于其客座编辑的身份，罗伯特-坦佩没有参与文章的同行评审，也无法获得有关同行评审的信息。这篇文章的编辑工作由西蒙-纽斯特德全权负责。

Declaration of competing interest
利益冲突声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有任何可能会影响本文所报告工作的已知经济利益或个人关系。

Data availability 数据可用性

No data was used for the research described in the article.
文章所述研究未使用任何数据。

Acknowledgments 致谢

We thank all members of the Institute of Biochemistry (Goethe University Frankfurt) for stimulating discussions. This research was supported the German Research Foundation (CRC 1507/P18 and CRC 1507/Z02 (No. 450648163), TA157/12-1 (Reinhard Koselleck Grant No. 407041508) and TA157/17-1 (No. 468346185) to R.T.) and the European Research Council (ERC Advanced Grant No. 789121 to R.T.).
我们感谢生物化学研究所（法兰克福歌德大学）的所有成员，感谢他们的热烈讨论。本研究得到了德国研究基金会（CRC 1507/P18 和 CRC 1507/Z02 (No. 450648163)，TA157/12-1 (Reinhard Koselleck Grant No. 407041508) 和 TA157/17-1 (No. 468346185) to R.T.）和欧洲研究理事会（ERC Advanced Grant No. 789121 to R.T.）的资助。

References 参考资料

Papers of particular interest, published within the period of review, have been highlighted as:
在审查期间发表的特别值得关注的论文有
  • of special interest 特别关注
    ∙ ∙ ∙\bullet of outstanding interest
    ∙ ∙ ∙\bullet 未偿利息
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    The authors present the first structure of the fully assembled BCR, consisting of the antigen-recognizing membrane-bound immunoglobulin ( mlg ) and the transmembrane signaling chains     lg ⁡ α lg ⁡ α lg alpha\lg \alpha and lg ⁡ β lg ⁡ β lg beta\lg \beta.
    作者首次展示了由抗原识别膜结合免疫球蛋白（mlg）、跨膜信号链 lg ⁡ α lg ⁡ α lg alpha\lg \alpha 和 lg ⁡ β lg ⁡ β lg beta\lg \beta 组成的完全组装 BCR 结构。
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    The authors present the first structure of the fully assembled BCR, consisting of the antigen-recognizing membrane-bound immunoglobulin ( mlg ) and the transmembrane signaling chains     lg ⁡ α lg ⁡ α lg alpha\lg \alpha and lg ⁡ β lg ⁡ β lg beta\lg \beta.
    作者首次展示了由抗原识别膜结合免疫球蛋白（mlg）、跨膜信号链 lg ⁡ α lg ⁡ α lg alpha\lg \alpha 和 lg ⁡ β lg ⁡ β lg beta\lg \beta 组成的完全组装 BCR 结构。
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