It takes two to signal The Hedgehog (HH) signaling pathway is important in development, and excessive HH signaling is associated with cancer. Signaling occurs through the G protein–coupled receptor Smoothened. The pathway is repressed by the membrane receptor Patched-1 (PTCH1), and this inhibition is relieved when PTCH1 binds the secreted protein HH. Two recent papers have described structures of HH bound to PTCH1, but surprisingly, each described a different binding epitope on HH. Qi et al. present a cryo–electron microscopy structure that explains this apparent contradiction by showing that a single HH protein uses both of these interfaces to engage two PTCH1 receptors (see the Perspective by Sommer and Lemmon). Functional assays suggest that both interfaces must be bound for efficient signaling. Science, this issue p. eaas8843; see also p. 26 The cryo–electron microscopy structure of a key complex involved in regulating a pathway important in development and cancer is elucidated. INTRODUCTION Appropriate Hedgehog (HH) signaling is important for human health; excessive HH signaling leads to various cancers, whereas insufficient HH signaling results in birth defects. The mature N-terminal domain of the HH protein with N-terminal palmitate and C-terminal cholesterol modifications (referred to as “native HH-N” here) binds to its receptor, Patched-1 (PTCH1), to release its repression of the HH pathway, presumably by regulating the transport of a ligand that regulates the downstream oncoprotein Smoothened. The palmitate moiety of HH-N is essential for HH signaling; however, the mechanism of how the palmitate moiety of HH induces the signaling is not clear. The means by which HH protein recognizes PTCH1 is also controversial: A recent 1:1 PTCH1–HH-N complex structure visualized a palmitate-mediated binding site on HH-N, which was inconsistent with previous studies that implied a distinct, Ca2+-mediated interface for PTCH1 and HH co-receptors. RATIONALE Cell biological studies indicated that mutations on either the palmitate-dominated interface or the Ca2+-mediated interface of HH-N eliminated signaling, indicating that both HH-N interfaces are necessary for HH to inhibit PTCH1 activity. We managed to assemble a 2:1 PTCH1–HH-N complex using a biologically functional PTCH1 variant (PTCH1*) and native Sonic HH-N (SHH-N) in the presence of 1 mM CaCl2 (a physiological extracellular calcium concentration) for structural investigation. RESULTS The cryo–electron microscopy (cryo-EM) structure of native SHH-N in complex with PTCH1* at 3.5-Å resolution demonstrates that one SHH-N molecule concomitantly engages both epitopes and binds two PTCH1 receptors (PTCH1-A and PTCH1-B) in an asymmetric manner. PTCH1-A and PTCH1-B exhibit several conformational differences and bind distinct portions of SHH-N. Structural comparisons suggest that the Ca2+-mediated interface of SHH-N physiologically binds different HH binders, including PTCH1, HH co-receptors, and the SHH-N antagonist 5E1 antibody, to regulate HH signaling. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces between SHH-N and PTCH1 is essential for maximal signaling in cells. Calculations using MOLE reveal a tunnel, ~150 Å in length with a minimum radius of 4 Å, that stretches through PTCH1-B but not PTCH1-A, which interacts with the palmitate of SHH-N. The path of this PTCH1-B tunnel includes the extracellular domain I (ECD-I), the cavity of the ECDs that accommodates the palmitate, and the transmembrane region. We introduced two point mutations (L427R and F1017E) on full-length human PTCH1 to block this tunnel by a ~3-Å salt bridge instead of by palmitate insertion. Cell biological assays show that unlike wild-type PTCH1, this variant was not able to repress the HH pathway; this finding supports our hypothesis that the palmitate can abolish the activity of PTCH1 by blocking the cavity of the ECDs. CONCLUSION Our structure of a signaling-competent complex consisting of SHH-N and an asymmetric arrangement of two PTCH1* molecules reconciles previous disparate findings of how HH-N interacts with PTCH1. The structural analysis reveals a tunnel in the PTCH1 molecule that can be blocked using mutations in the ECDs that mimic the palmitate insertion, thus abolishing HH pathway repression by PTCH1. Thus, our work illuminates how two sites on SHH-N serve to unite two PTCH1 receptors, thereby initiating a full HH signal. Cryo-EM structure of 2:1 human PTCH1*–SHH-N signaling-competent complex. PTCH1-A, PTCH1-B, and SHH-N are colored yellow, light blue, and cyan, respectively, and are viewed from the side of the membrane. Palmitate (PLM) is shown as magenta sticks, calcium as green spheres. Cholesterol in the cell membrane is shown as ball-and-stick models colored yellow and red. Aberrant Hedgehog (HH) signaling leads to various types of cancer and birth defects. N-terminally palmitoylated HH initiates signaling by binding its receptor Patched-1 (PTCH1). A recent 1:1 PTCH1-HH complex structure visualized a palmitate-mediated binding site on HH, which was inconsistent with previous studies that implied a distinct, calcium-mediated binding site for PTCH1 and HH co-receptors. Our 3.5-angstrom resolution cryo–electron microscopy structure of native Sonic Hedgehog (SHH-N) in complex with PTCH1 at a physiological calcium concentration reconciles these disparate findings and demonstrates that one SHH-N molecule engages both epitopes to bind two PTCH1 receptors in an asymmetric manner. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces is required for efficient signaling in cells.

中文翻译：

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两个被修补的分子与 Hedgehog 上的不同位点结合，产生信号传导复合物

需要两个信号才能发出信号 Hedgehog (HH) 信号通路在发育过程中很重要，而过度的 HH 信号通路与癌症有关。信号通过 G 蛋白偶联受体平滑。该通路受到膜受体 Patched-1 (PTCH1) 的抑制，当 PTCH1 与分泌蛋白 HH 结合时，这种抑制作用就会解除。最近的两篇论文描述了 HH 与 PTCH1 结合的结构，但令人惊讶的是，每篇论文都描述了 HH 上不同的结合表位。齐等人。提出了一种冷冻电子显微镜结构，通过显示单个 HH 蛋白使用这两个界面来接合两个 PTCH1 受体来解释这一明显的矛盾（参见 Sommer 和 Lemmon 的观点）。功能分析表明，必须绑定两个接口才能有效地发出信号。科学，这个问题 p。eaas8843; 另见第。26 阐明了参与调节在发育和癌症中重要的途径的关键复合物的冷冻电子显微镜结构。引言 适当的刺猬 (HH) 信号对人类健康很重要。过多的 HH 信号会导致各种癌症，而不足的 HH 信号会导致出生缺陷。具有 N 端棕榈酸酯和 C 端胆固醇修饰的 HH 蛋白的成熟 N 端结构域（此处称为“天然 HH-N”）与其受体 Patched-1 (PTCH1) 结合，以释放其对HH 途径，大概是通过调节配体的转运来调节下游癌蛋白 Smoothened。HH-N 的棕榈酸酯部分对 HH 信号传导至关重要；然而，HH 的棕榈酸酯部分如何诱导信号传导的机制尚不清楚。HH 蛋白识别 PTCH1 的方式也存在争议：最近的 1:1 PTCH1-HH-N 复合结构显示了 HH-N 上棕榈酸酯介导的结合位点，这与之前的研究不一致，该研究暗示了一个独特的、Ca2+ 介导的PTCH1 和 HH 共同受体的接口。基本原理 细胞生物学研究表明，HH-N 的棕榈酸酯占主导地位的界面或 Ca2+ 介导的界面上的突变消除了信号传导，表明这两个 HH-N 界面都是 HH 抑制 PTCH1 活性所必需的。我们设法在存在 1 mM CaCl2（生理细胞外钙浓度）的情况下，使用具有生物学功能的 PTCH1 变体 (PTCH1*) 和天然 Sonic HH-N (SHH-N) 组装了一个 2:1 PTCH1–HH-N 复合物，用于结构调查。结果 天然 SHH-N 与 PTCH1* 复合物在 3.5 Å 分辨率下的冷冻电子显微镜 (cryo-EM) 结构表明，一个 SHH-N 分子同时结合两个表位并结合两个 PTCH1 受体（PTCH1-A 和 PTCH1- B) 以不对称的方式。PTCH1-A 和 PTCH1-B 表现出几种构象差异并结合 SHH-N 的不同部分。结构比较表明，Ca2+ 介导的 SHH-N 界面在生理上结合不同的 HH 结合剂，包括 PTCH1、HH 共受体和 SHH-N 拮抗剂 5E1 抗体，以调节 HH 信号传导。使用 PTCH1 或 SHH-N 突变体破坏单个界面的功能分析表明，SHH-N 和 PTCH1 之间的两个界面的同时接合对于细胞中的最大信号传导至关重要。使用 MOLE 的计算揭示了一个隧道，长度约为 150 Å，最小半径为 4 Å，它穿过 PTCH1-B，但不穿过 PTCH1-A，后者与 SHH-N 的棕榈酸酯相互作用。此 PTCH1-B 隧道的路径包括胞外域 I (ECD-I)、容纳棕榈酸酯的 ECD 腔和跨膜区。我们在全长人类 PTCH1 上引入了两个点突变（L427R 和 F1017E），以通过 ~3-Å 盐桥而不是通过棕榈酸酯插入来阻断该隧道。细胞生物学分析表明，与野生型 PTCH1 不同，该变体不能抑制 HH 通路；这一发现支持了我们的假设，即棕榈酸酯可以通过阻断 ECD 的腔来消除 PTCH1 的活性。结论 我们的信号传导复合物的结构由 SHH-N 和两个 PTCH1* 分子的不对称排列组成，与之前关于 HH-N 如何与 PTCH1 相互作用的不同发现相一致。结构分析揭示了 PTCH1 分子中的一个隧道，可以使用模拟棕榈酸插入的 ECD 中的突变来阻断该隧道，从而消除 PTCH1 对 HH 通路的抑制。因此，我们的工作阐明了 SHH-N 上的两个位点如何结合两个 PTCH1 受体，从而启动完整的 HH 信号。2:1 人类 PTCH1*–SHH-N 信号传导复合物的冷冻电镜结构。PTCH1-A、PTCH1-B 和 SHH-N 分别为黄色、浅蓝色和青色，从膜的侧面观察。棕榈酸酯 (PLM) 显示为洋红色棒，钙显示为绿色球体。细胞膜中的胆固醇显示为黄色和红色的球棒模型。异常刺猬 (HH) 信号会导致各种类型的癌症和先天缺陷。N-末端棕榈酰化的 HH 通过结合其受体 Patched-1 (PTCH1) 启动信号传导。最近的 1:1 PTCH1-HH 复合结构显示了 HH 上棕榈酸酯介导的结合位点，这与之前的研究不一致，后者暗示 PTCH1 和 HH 共受体有一个独特的钙介导的结合位点。我们在生理钙浓度下与 PTCH1 复合的天然 Sonic Hedgehog (SHH-N) 的 3.5 埃分辨率冷冻电子显微镜结构协调了这些不同的发现，并证明一个 SHH-N 分子与两个表位结合，以结合两个 PTCH1 受体不对称方式。