São Carlos Institute of Chemistry, University of São Paulo, São Carlos, SP, Brazil.
São Carlos Institute of Chemistry, University of São Paulo, São Carlos, SP, Brazil; Exact and Technology Sciences Center, Federal University of São Carlos, São Carlos, SP 13560-970, Brazil.
Biochim Biophys Acta Proteins Proteom. 2024 Sep 1;1872(5):141034. doi: 10.1016/j.bbapap.2024.141034. Epub 2024 Jul 14.
The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.
HSPA5 蛋白（BiP/Grp78）作为维持细胞蛋白质质量控制的关键伴侣。作为人类 HSP70 家族的一员，HSPA5 由两个不同的结构域组成：核苷酸结合域（NBD）和肽结合域（PBD）。在这项研究中，我们研究了 HSPA5 的结构域间相互作用，旨在阐明这些结构域如何调节其作为伴侣的功能。我们的研究结果表明，全长 HSPA5（HSPA5-FL）、截短的 HSPA5-T 和 HSPA5-N 对 ATP 和 ADP 具有不同的亲和力，对镁的依赖性很大，以保证最佳的相互作用。有趣的是，在 ADP 实验中，金属离子的存在似乎仅增强了 HSPA5-FL 和 HSPA5-T 的 NBD 结合。此外，虽然 C 端截短不会显著影响 HSPA5 的热稳定性，但涉及 MgATP 的实验强调了其在介导相互作用和核苷酸水解中的重要作用。热稳定性实验进一步表明，NBD-PBD 界面增强了 NBD 的稳定性，对 HSPA5 的影响比同源的 HSPA1A 更明显，通过结构域间的偶联防止了自身聚集。酶学分析表明，PBD 的存在增强了 NBD 的 ATP 酶活性并增加了其核苷酸亲和力。值得注意的是，PBD 的内在伴侣活性依赖于 NBD 的存在，这可能是由于 PBD 自身寡聚化的倾向。总的来说，我们的数据强调了变构机制在调节 HSPA5 的热稳定性、核苷酸相互作用和 ATP 酶活性方面的关键作用，突出了其在细胞环境中蛋白质质量控制中的重要性。
