Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2013 May 21;110(21):8513-8. doi: 10.1073/pnas.1217988110. Epub 2013 May 6.
Heat shock protein (Hsp) 104 is a ring-forming, protein-remodeling machine that harnesses the energy of ATP binding and hydrolysis to drive protein disaggregation. Although Hsp104 is an active ATPase, the recovery of functional protein requires the species-specific cooperation of the Hsp70 system. However, like Hsp104, Hsp70 is an active ATPase, which recognizes aggregated and aggregation-prone proteins, making it difficult to differentiate the mechanistic roles of Hsp104 and Hsp70 during protein disaggregation. Mapping the Hsp70-binding sites in yeast Hsp104 using peptide array technology and photo-cross-linking revealed a striking conservation of the primary Hsp70-binding motifs on the Hsp104 middle-domain across species, despite lack of sequence identity. Remarkably, inserting a Strep-Tactin binding motif at the spatially conserved Hsp70-binding site elicits the Hsp104 protein disaggregating activity that now depends on Strep-Tactin but no longer requires Hsp70/40. Consistent with a Strep-Tactin-dependent activation step, we found that full-length Hsp70 on its own could activate the Hsp104 hexamer by promoting intersubunit coordination, suggesting that Hsp70 is an activator of the Hsp104 motor.
热休克蛋白 104（Hsp104）是一种环形形成的蛋白质重塑机器，它利用 ATP 结合和解离的能量来驱动蛋白质解聚。尽管 Hsp104 是一种活性 ATP 酶，但功能蛋白的恢复需要特定物种的 Hsp70 系统的特异性合作。然而，与 Hsp104 一样，Hsp70 也是一种活性 ATP 酶，它能识别聚集和易聚集的蛋白质，这使得难以区分 Hsp104 和 Hsp70 在蛋白质解聚过程中的机制作用。使用肽阵列技术和光交联技术在酵母 Hsp104 上定位 Hsp70 结合位点，揭示了尽管缺乏序列同一性，但在不同物种中 Hsp104 中间域上的主要 Hsp70 结合基序具有惊人的保守性。值得注意的是，在空间保守的 Hsp70 结合位点插入 Strep-Tactin 结合基序会引发 Hsp104 蛋白解聚活性，该活性现在依赖于 Strep-Tactin，但不再需要 Hsp70/40。与 Strep-Tactin 依赖性激活步骤一致，我们发现全长 Hsp70 本身可以通过促进亚基间协调来激活 Hsp104 六聚体，这表明 Hsp70 是 Hsp104 马达的激活剂。
