Human Aha1's N-terminal extension confers it holdase activity in vitro
Human Aha1's N-terminal extension confers it holdase activity in vitro
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DOI：
10.1002/pro.4735
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发表时间：
2023-09-01
期刊：
影响因子：
8
通讯作者：
Zhang，Naixia
中科院分区：
文献类型：
--
作者：
Tang，Junying;Hu，Huifang;Zhang，Naixia
分子伴侣是蛋白质质量控制系统的关键组成部分，在控制蛋白质稳态中发挥着重要作用。 Aha1 已被确定为 Hsp90 的辅助伴侣，可强烈加速 Hsp90 的 ATP 酶活性。同时，据报道，Aha1 还可以充当自主伴侣，保护应激或无序的蛋白质免于聚集。本文进行了一系列体外实验来验证 Aha1 是否具有非 Hsp90 依赖性的保持酶活性，并阐明底物识别的相关分子机制。根据重折叠测定的结果，来自高等真核生物的 Aha1 中跨越 M1 至 R16 的高度保守的 N 端延伸负责该蛋白质的保持酶活性。 NMR数据显示，Aha1的N端延伸在溶液中主要采用无序构象，与Aha1 N端结构域的核心结构没有紧密接触。基于本质上无序的结构特征和 Aha1 N 末端延伸的一级序列，预期涉及该特定区域和未折叠底物蛋白的模糊型蛋白质-蛋白质相互作用。以下突变分析数据表明，可能涉及包括 W4 和 W11 在内的两种色氨酸的范德华接触在 Aha1 和未折叠麦芽糖结合蛋白 (MBP) 之间的相互作用中不起主导作用。同时，由于高浓度的 NaCl 可以消除 Aha1 的保持酶活性，因此 Aha1 N 末端延伸中的带电残基介导的静电相互作用在底物识别中发挥着至关重要的作用。
Molecular chaperones are key components of protein quality control system, which plays an essential role in controlling protein homeostasis. Aha1 has been identified as a co‐chaperone of Hsp90 known to strongly accelerate Hsp90's ATPase activity. Meanwhile, it is reported that Aha1 could also act as an autonomous chaperone and protect stressed or disordered proteins from aggregation. Here, in this article, a series of in vitro experiments were conducted to verify whether Aha1 has a non‐Hsp90‐dependent holdase activity and to elucidate the associated molecular mechanism for substrate recognition. According to the results of the refolding assay, the highly conserved N‐terminal extension spanning M1 to R16 in Aha1 from higher eukaryotes is responsible for the holdase activity of the protein. As revealed by the NMR data, Aha1's N‐terminal extension mainly adopts a disordered conformation in solution and shows no tight contacts with the core structure of Aha1's N‐terminal domain. Based on the intrinsically disordered structure feature and the primary sequence of Aha1's N‐terminal extension, the fuzzy‐type protein–protein interactions involving this specific region and the unfolded substrate proteins are expected. The following mutation analysis data demonstrated that the Van der Waals contacts potentially involving two tryptophans including W4 and W11 do not play a dominant role in the interaction between Aha1 and unfolded maltose binding protein (MBP). Meanwhile, since the high concentration of NaCl could abolish the holdase activity of Aha1, the electrostatic interactions mediated by those charged residues in Aha1's N‐terminal extension are thus indicated to play a crucial role in the substrate recognition.