From the National Laboratory of Biomacromolecules, Chinese Academy of Sciences Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
From the National Laboratory of Biomacromolecules, Chinese Academy of Sciences Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of the Chinese Academy of Sciences, Beijing 100049, China.
J Biol Chem. 2018 Nov 16;293(46):17663-17675. doi: 10.1074/jbc.RA118.002691. Epub 2018 Sep 18.
The allosteric coupling of the highly conserved nucleotide- and substrate-binding domains of Hsp70 has been studied intensively. In contrast, the role of the disordered, highly variable C-terminal region of Hsp70 remains unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD motif binds to the tetratricopeptide-repeat domains of Hsp70 co-chaperones. Here, we discovered that the TVEEVD sequence of cytoplasmic Hsp70 (Ssa1) functions as a SUMO-interacting motif. A second C-terminal motif of ∼15 amino acids between the α-helical lid and the extreme C terminus, previously identified in bacterial and eukaryotic organellar Hsp70s, is known to enhance chaperone function by transiently interacting with folding clients. Using structural analysis, interaction studies, fibril formation assays, and functional assays, we investigated the individual contributions of the α-helical bundle and the C-terminal disordered region of Ssa1 in the inhibition of fibril formation of the prion protein Ure2. Our results revealed that although the α-helical bundle of the Ssa1 substrate-binding domain (SBDα) does not directly bind to Ure2, the SBDα enhances the ability of Hsp70 to inhibit fibril formation. We found that a 20-residue C-terminal motif in Ssa1, containing GGAP and GGAP-like tetrapeptide repeats, can directly bind to Ure2, the Hsp40 co-chaperone Ydj1, and α-synuclein, but not to the SUMO-like protein SMT3 or BSA. Deletion or substitution of the Ssa1 GGAP motif impaired yeast cell tolerance to temperature and cell-wall damage stress. This study highlights that the C-terminal GGAP motif of Hsp70 is important for substrate recognition and mediation of the heat shock response.
高度保守的核苷酸和底物结合域的变构偶联在 Hsp70 中已被深入研究。相比之下，Hsp70 无规则、高度可变的 C 端区域的作用仍不清楚。在许多真核 Hsp70 中，极端 C 端 EEVD 基序与 Hsp70 共伴侣的四肽重复结构域结合。在这里，我们发现细胞质 Hsp70（Ssa1）的 TVEEVD 序列作为 SUMO 相互作用基序。以前在细菌和真核细胞器 Hsp70 中发现的位于 α-螺旋盖和极端 C 端之间的约 15 个氨基酸的第二个 C 端基序，已知通过与折叠的客户短暂相互作用来增强伴侣的功能。使用结构分析、相互作用研究、纤维形成测定和功能测定，我们研究了 Ssa1 的α-螺旋束和 C 端无规区在抑制 Ure2 朊病毒蛋白纤维形成中的个体贡献。我们的结果表明，尽管 Ssa1 的底物结合域（SBDα）的α-螺旋束不直接与 Ure2 结合，但 SBDα增强了 Hsp70 抑制纤维形成的能力。我们发现，Ssa1 中包含 GGAP 和 GGAP 样四肽重复的 20 个残基 C 端基序，可以直接与 Ure2、Hsp40 共伴侣 Ydj1 和α-突触核蛋白结合，但不能与 SUMO 样蛋白 SMT3 或 BSA 结合。Ssa1 GGAP 基序的缺失或取代会损害酵母细胞对温度和细胞壁损伤应激的耐受性。本研究强调了 Hsp70 的 C 端 GGAP 基序在底物识别和介导热休克反应中的重要性。
