From the Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts 01003.
Department of Chemistry, Physical & Theoretical Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, United Kingdom, and.
J Biol Chem. 2018 Dec 21;293(51):19511-19521. doi: 10.1074/jbc.RA118.005421. Epub 2018 Oct 22.
Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (Ta and Ps) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the Ta and Ps covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Ta (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp-substrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.
小分子热休克蛋白（sHsps）是普遍存在的分子伴侣，sHsp 的突变或表达改变与多种人类疾病状态有关。sHsp 单体组装成具有二聚体亚结构的大型寡聚物，sHsp 寡聚物的动力学导致了关于捕获底物的形式的重大问题，这是其作用机制的关键方面。我们在这里表明，两种植物十二聚体 sHsps（Ta16.9 和同源 Ps18.1）的亚结构二聚体是与展开底物最初相遇时的功能单位。我们在两个十二聚体界面（二聚体和非二聚体）引入了多肽间二硫键，以限制其组装体的解离方式。当在非二聚体界面上形成二硫键时，Ta16.9 和 Ps18.1 的突变体（Ta 和 Ps）失活，但还原后具有 WT 样伴侣活性，表明非二聚体界面的解离对于 sHsp 活性是必不可少的。此外，Ta 和 Ps 共价单元的大小为这些 sHsps 定义了一个新的四面体几何形状，与 Ta16.9 X 射线结构中观察到的不同。重要的是，氧化 Ta（在二聚体界面上形成二硫键）表现出大大增强的保护底物的能力，表明增强二聚体界面可以提高伴侣效率。温度诱导的大小和二级结构变化表明折叠的 sHsp 二聚体与底物相互作用，并且二聚体稳定性影响伴侣效率。这些结果提供了一个模型，其中 sHsp 二聚体在组装成更大、异质的 sHsp-底物复合物之前捕获底物，用于底物重折叠或降解，并且表明调节二聚体界面的强度可以用于设计 sHsp 伴侣效率。
