Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway.
Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway.
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DOI：
10.1038/s42003-023-05579-3
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发表时间：
2023-12-18
影响因子：
5.9
通讯作者：
Drennan, Catherine L.
中科院分区：
文献类型：
--
作者：
Vasquez, Sheena;Marquez, Melissa D.;Brignole, Edward J.;Vo, Amanda;Kong, Sunnie;Park, Christopher;Perlstein, Deborah L.;Drennan, Catherine L.
铁硫团簇对生命至关重要，其生物合成缺陷导致人类疾病。通过胞质铁硫簇组装（CIA）途径，簇组装和传递到细胞质和核客户蛋白的机制尚不清楚。在这里，我们报告了来自酿酒酵母的热重复蛋白Met18的低温电镜结构，这是CIA靶向复合物（CTC）的关键组成部分，可以识别细胞质和核客户蛋白并提供成熟的铁硫簇。我们发现在没有其他CTC蛋白的情况下，Met18呈四聚体和六聚体状态。通过质谱法和负染色电镜，我们发现在加入Cia2后，这些高阶低聚态的Met18被分解。我们还使用下拉法来鉴定对Cia2结合和Leu1客户端识别至关重要的残基，其中许多残基在Met18寡聚时被掩埋。我们的结构显示了Met18的构象，这是以前在任何Met18同源物中都没有观察到的，这支持了这样一种观点，即高度柔性的Met18可能是CTC如何能够将铁硫簇传递给各种大小和形状的客户蛋白的关键，即Met18符合所需的尺寸。对CIA Pathway热重复蛋白Met18的结构和生化研究表明，Met18形成不同的低聚物，具有结合客户蛋白Leu1的柔性n端和结合CIA蛋白Cia2的c端。
Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed. Structural and biochemical studies of Met18, a CIA Pathway heat repeat protein, show that Met18 forms different oligomers, has a flexible N-terminus that binds client protein Leu1, and a C-terminus that binds CIA protein Cia2.
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影响因子：
16.8
作者：
Farnung L;Ochmann M;Engeholm M;Cramer P
通讯作者：
Cramer P
DOI：
10.1126/science.1219723
发表时间：
2012-07-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Stehling O;Vashisht AA;Mascarenhas J;Jonsson ZO;Sharma T;Netz DJ;Pierik AJ;Wohlschlegel JA;Lill R
通讯作者：
Lill R
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.8
作者：
van Wietmarschen, Niek;Moradian, Annie;Uringa, Evert-Jan
通讯作者：
Uringa, Evert-Jan
影响因子：
6.6
作者：
Lill, Roland;Dutkiewicz, Rafal;Milhlenhoff, Ulrich
通讯作者：
Milhlenhoff, Ulrich