Program in Biochemistry, Mount Holyoke College, South Hadley, MA 01075, USA.
Program in Neuroscience and Behavior, Mount Holyoke College, South Hadley, MA 01075, USA.
Int J Mol Sci. 2023 Dec 29;25(1):471. doi: 10.3390/ijms25010471.
Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that function as "holdases" and prevent protein aggregation due to changes in temperature, pH, or oxidation state. sHsps have a conserved α-crystallin domain (ACD), which forms the dimer building block, flanked by variable N- and C-terminal regions. sHsps populate various oligomeric states as a function of their sequestrase activity, and these dynamic structural features allow the proteins to interact with a plethora of cellular substrates. However, the molecular mechanisms of their dynamic conformational assembly and the interactions with various substrates remains unclear. Therefore, it is important to gain insight into the underlying physicochemical properties that influence sHsp structure in an effort to understand their mechanism(s) of action. We evaluated several disease-relevant mutations, D109A, F113Y, R116C, R120G, and R120C, in the ACD of HspB5 for changes to in vitro chaperone activity relative to that of wildtype. Structural characteristics were also evaluated by ANS fluorescence and CD spectroscopy. Our results indicated that mutation Y113F is an efficient holdase, while D109A and R120G, which are found in patients with myofibrillar myopathy and cataracts, respectively, exhibit a large reduction in holdase activity in a chaperone-like light-scattering assay, which indicated alterations in substrate-sHsp interactions. The extent of the reductions in chaperone activities are different among the mutants and specific to the substrate protein, suggesting that while sHsps are able to interact with many substrates, specific interactions provide selectivity for some substrates compared to others. This work is consistent with a model for chaperone activity where key electrostatic interactions in the sHsp dimer provide structural stability and influence both higher-order sHsp interactions and facilitate interactions with substrate proteins that define chaperone holdase activity.
小分子热休克蛋白（sHsps）是一组 ATP 非依赖性分子伴侣，作为“持家蛋白”发挥作用，防止由于温度、pH 值或氧化状态的变化导致蛋白质聚集。sHsps 具有保守的α-晶体蛋白结构域（ACD），形成二聚体构建块，两侧是可变的 N-和 C-末端区域。sHsps 作为其隔离酶活性的功能，存在于各种寡聚状态，这些动态结构特征允许蛋白质与大量细胞底物相互作用。然而，其动态构象组装的分子机制以及与各种底物的相互作用仍然不清楚。因此，深入了解影响 sHsp 结构的基础理化性质对于理解其作用机制非常重要。我们评估了 ACD 中与几种疾病相关的突变，包括 D109A、F113Y、R116C、R120G 和 R120C，以了解相对于野生型，它们对体外伴侣活性的影响。还通过 ANS 荧光和 CD 光谱学评估了结构特征。我们的结果表明，突变 Y113F 是一种有效的持家蛋白，而 D109A 和 R120G，分别存在于肌原纤维肌病和白内障患者中，在伴侣样光散射测定中显示出持家酶活性的大幅降低，这表明底物-sHsp 相互作用发生了改变。突变体之间的伴侣酶活性降低程度不同，并且对特定的底物蛋白具有特异性，这表明虽然 sHsps 能够与许多底物相互作用，但特定的相互作用为某些底物相对于其他底物提供了选择性。这项工作与伴侣酶活性的模型一致，其中 sHsp 二聚体中的关键静电相互作用提供结构稳定性，并影响 sHsp 的高级相互作用，以及促进与定义伴侣酶持家酶活性的底物蛋白的相互作用。
