Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104
Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104
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DOI：
10.1074/jbc.m403777200
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发表时间：
2004-07-09
影响因子：
4.8
通讯作者：
Glover, JR
中科院分区：
文献类型：
--
作者：
Lum, R;Tkach, JM;Glover, JR
酿酒酵母Hsp104是AAA+ATPase Hsp100/CLP亚家族中的一个六聚体成员，具有两个核苷酸结合域(NBD1和2)，它与Hsp70分子伴侣一起折叠聚集的蛋白质。HSP104可以作为一个“分子撬棍”撬开聚集体和/或可以通过展开和穿过HSP104六聚体的轴向通道来从聚集体中提取蛋白质。针对Tyr-662，位于Nbd2中轴向通道环的Gly-Tyr-Val-Gly基序中，我们创建了保守的(Phe和Trp)和非保守的(Ala和Lys)氨基酸替换。这些HSP104衍生物中的每一个都可以与野生型蛋白相媲美，因为它们能够水解三磷酸腺苷，组装成六聚体，并与热休克诱导的活细胞聚集体结合。然而，只有那些具有保守替换的基因才能弥补Deltahsp104酵母菌株的耐热性缺陷，并在体外促进聚集蛋白的复性。监测Trp-662的荧光显示，用ATP或ADP滴定完全组装的分子会逐渐猝灭荧光，这表明核苷酸结合决定了环在轴向通道中的位置。Nbd2轴通道中645位的Glu到Lys的替换强烈地改变了核苷酸诱导的Trp-662的荧光变化，并特异性地损害了蛋白质的折叠。这些数据表明，通过Nbd2的轴向通道的结构完整性是Hsp104发挥作用所必需的，并支持Hsp104和ClpB使用类似的展开/穿线机制来促进解聚和重折叠的提议，而其他Hsp100则使用这种机制来促进蛋白质降解。
Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.