Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity. To address the mechanism of the differences in cysteine reactivity between hHsp70 and hHsc70, we studied the factors that determine this reactivity by Ellman assay for the quantification of accessible free thiols and NMR analysis for the assessment of structural dynamics. We found the lower cysteine reactivity of hHsc70 is probably due to its lower structural dynamics and the stronger inhibition effect of interaction between the α-helical lid subdomain of the substrate-binding domain (SBDα) and the β-sheet substrate-binding subdomain (SBDβ) on cysteine reactivity of hHsc70. We determined that Gly557 in hHsp70 contributes significantly to the higher structural dynamics and cysteine reactivity of hHsp70 SBDα. Exploring the cysteine reactivity of hHsp70 and hHsc70 facilitates an understanding of the effects of redox reactions and electrophiles on their chaperone activity and regulation mechanisms, and how these differences allow them to undertake distinct cellular roles.

Hsp70 是多功能蛋白质，是蛋白质质量控​​制网络的中心枢纽。Hsp70 还与多种疾病相关，已被确定为药物靶点。人 HspA1A (hHsp70) 和 HspA8 (hHsc70) 是主要的细胞溶质 Hsp70，它们具有重叠和不同的功能。hHsp70 包含五个半胱氨酸残基，hHsc70 包含四个半胱氨酸残基。先前的研究表明这些半胱氨酸残基可以进行不同的半胱氨酸修饰，例如氧化或与亲电试剂反应以调节其功能，并且hHsp70和hHsc70具有不同的半胱氨酸反应性。为了解决 hHsp70 和 hHsc70 之间半胱氨酸反应性差异的机制，我们通过 Ellman 测定法研究了确定这种反应性的因素，以量化可获得的游离硫醇和 NMR 分析以评估结构动力学。我们发现 hHsc70 较低的半胱氨酸反应性可能是由于其较低的结构动力学和底物结合结构域 (SBDα) 的 α-螺旋盖亚结构域 (SBDα) 与 β-折叠底物结合亚结构域 (SBDβ) 之间相互作用的更强抑制作用) 对 hHsc70 的半胱氨酸反应性。我们确定 hHsp70 中的 Gly557 对 hHsp70 SBDα 的更高结构动力学和半胱氨酸反应性有显着贡献。探索 hHsp70 和 hHsc70 的半胱氨酸反应性有助于了解氧化还原反应和亲电子试剂对其伴侣活性和调节机制的影响，