Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; Julius Clinical Ltd, Broederplein 41-43, 3703 CD Zeist, the Netherlands(‡).
J Mol Biol. 2021 Jun 25;433(13):166955. doi: 10.1016/j.jmb.2021.166955. Epub 2021 Mar 24.
ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.
ABC 转运蛋白跨膜运输多种分子，由跨膜和胞质结构域组成。它们的活性循环涉及到严格调控和协调的结构域协同作用。调节由胞质结构域驱动，功能由跨膜结构域执行。这些多结构域的多拓扑蛋白折叠成其功能状态是细胞面临的挑战，共翻译和顺序事件缓解了这一挑战。我们在此揭示了 CFTR 的共翻译结构域折叠和组装的最初阶段，CFTR 是最常见的罕见遗传性疾病囊性纤维化中 ABC 转运蛋白缺陷的原因。我们结合了生物合成放射性标记与蛋白酶敏感性测定和结构域特异性抗体。最 N 端结构域 TMD1（跨膜结构域 1）在翻译过程中折叠其疏水性和可溶性螺旋：跨膜螺旋紧密包装，胞质 N 和 C 末端与第一个胞质螺旋环 ICL1 组装，仅留下 ICL2 暴露在外。这种 N-C-ICL1 组装通过两个独立事件得到加强：（i）ICL1 与下一个结构域胞质 NBD1（核苷酸结合结构域 1）的 N 端亚结构域组装；和（ii）在校正药物 VX-809 的存在下，该药物可挽救一系列致病 CFTR 突变体的细胞表面表达。这两种情况都增加了 CFTR N 端的屏蔽，其叠加性意味着不同的作用模式。NBD1 和 TMD1 的早期组装对于 CFTR 折叠至关重要，并将两个结构域定位在与 TMD2 所需的组装位置。总的来说，我们已经深入了解了 CFTR 翻译过程中和翻译后立即发生的这第一个、核形成的、VX-809 增强的结构域组装事件，涉及到 I 型 ABC 外排泵中保守的结构。
