Structure-function analysis of the heme-binding WWD domain in the bacterial holocytochrome c synthase, CcmFH.
Structure-function analysis of the heme-binding WWD domain in the bacterial holocytochrome c synthase, CcmFH.
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DOI：
10.1128/mbio.01509-23
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发表时间：
2023-12-19
期刊：
影响因子：
6.4
通讯作者：
Sutherland, Molly C.
中科院分区：
文献类型：
--
作者：
Grunow, Amber L.;Carroll, Susan C.;Kreiman, Alicia N.;Sutherland, Molly C.
血红素运输是一个基本的生物过程，但由于血红素严格的细胞内调节、血红素细胞毒性和运输的短暂性，其直接研究受到阻碍。细菌系统 I 和系统 II 细胞色素 c 生物发生途径正在发展成为研究血红素运输机制的模型，因为它们的功能是将血红素从细胞内部运输到细胞外部以附着到脱辅基细胞色素 c。细胞色素 c 需要血红素进行折叠，并在电子传递链中发挥作用，以实现关键的细胞功能，例如呼吸。我们重点关注系统 I，它由八种膜蛋白 CcmABCDEFGH 组成，建议分两步发挥作用：CcmABCD 介导血红素的转移和与 CcmE 的附着。 HoloCcmE 将血红素陪伴至 CcmFH，以附着脱辅基细胞色素 c。虽然已知 CcmFH 是全细胞色素 c 合酶，但血红素相互作用和与脱辅基细胞色素 c 结合的定位机制仍有待阐明。利用丙氨酸扫描和半胱氨酸扫描对 CcmF 中保守的 WWD 结构域进行了全面的结构功能分析，揭示了对 CcmF 合酶功能至关重要的残基以及与血红素 2- 和 4- 乙烯基相互作用所需的残基。该分析首次证明 CcmF WWD 结构域直接与血红素相互作用，并且该结构域内的血红素相互作用是附着脱辅基细胞色素 c 所必需的。这种对血红素结合的深入研究现在可以对细胞色素 c 生物发生蛋白 CcmF、CcmC 和 CcsBA 进行比较，揭示这些血红素运输途径中血红素相互作用的常见机制。血红素是参与关键细胞功能（例如能量产生和氧气运输）的蛋白质的重要辅助因子。因此，了解血红素如何与蛋白质相互作用以及如何在细胞中移动是一个基本的生物学问题。这项工作研究了系统 I 细胞色素 c 生物发生途径，在某些物种（包括大肠杆菌）中，该途径由八种称为 CcmA-H 的完整膜或膜相关蛋白组成，这些蛋白被认为分两个步骤发挥作用，将血红素转运和附着到脱辅基细胞色素 c。细胞色素 c 需要这种血红素附着物才能在电子传递链中发挥作用，以产生细胞能量。分析了 CcmFH 中保守的 WWD 血红素处理结构域，并鉴定了对血红素相互作用和全细胞色素 c 合酶活性至关重要的残基。 CcmFH 是包含 WWD 结构域的血红素处理蛋白家族的第三个成员，经过全面的结构功能分析，可以比较该蛋白家族中血红素的相互作用。
Heme trafficking is a fundamental biological process, yet its direct study has been hampered due to heme’s tight intracellular regulation, heme cytotoxicity, and the transient nature of trafficking. The bacterial System I and System II cytochrome c biogenesis pathways are developing into models to interrogate heme trafficking mechanisms, as they function to transport heme from inside to outside the cell for attachment to apocytochrome c. Cytochromes c require heme for folding and to function in the context of electron transport chains for critical cellular functions, such as respiration. We focus on System I, comprised of eight membrane proteins, CcmABCDEFGH, proposed to function in two steps: CcmABCD mediates the transfer of heme and attachment to CcmE. HoloCcmE chaperones heme to CcmFH for attachment to apocytochrome c. While CcmFH is known to be the holocytochrome c synthase, the mechanism of heme interaction and positioning for attachment to apocytochrome c remains to be elucidated. A comprehensive structure-function analysis of the conserved WWD domain in CcmF was undertaken utilizing alanine-scanning and cysteine-scanning, revealing residues critical for CcmF’s synthase function and residues required for interaction with the 2- and 4-vinyls of heme. This analysis demonstrates for the first time that the CcmF WWD domain directly interacts with heme and that heme interactions within this domain are required for attachment to apocytochrome c. This in-depth interrogation of heme binding now allows for comparison across cytochrome c biogenesis proteins CcmF, CcmC, and CcsBA, revealing common mechanisms of heme interaction in these heme trafficking pathways. Heme is an essential co-factor for proteins involved with critical cellular functions, such as energy production and oxygen transport. Thus, understanding how heme interacts with proteins and is moved through cells is a fundamental biological question. This work studies the System I cytochrome c biogenesis pathway, which in some species (including Escherichia coli) is composed of eight integral membrane or membrane-associated proteins called CcmA-H that are proposed to function in two steps to transport and attach heme to apocytochrome c. Cytochrome c requires this heme attachment to function in electron transport chains to generate cellular energy. A conserved WWD heme-handling domain in CcmFH is analyzed and residues critical for heme interaction and holocytochrome c synthase activity are identified. CcmFH is the third member of the WWD domain-containing heme-handling protein family to undergo a comprehensive structure-function analysis, allowing for comparison of heme interaction across this protein family.
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影响因子：
16.6
作者：
Ilcu, Lorena;Denkhaus, Lukas;Brausemann, Anton;Zhang, Lin;Einsle, Oliver
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DOI：
10.1073/pnas.1615195114
发表时间：
2017-03-14
影响因子：
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