Intercollegiate Faculty of Biotechnology UG-MUG, University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland.
Magnetic Resonance Center and Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019 Sesto Fiorentino, Italy.
J Mol Biol. 2021 Jul 23;433(15):167054. doi: 10.1016/j.jmb.2021.167054. Epub 2021 May 20.
Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure.
小分子热休克蛋白 (sHsps) 是一类保守的 ATP 非依赖性伴侣蛋白，在应激条件下与未折叠的蛋白质底物结合，防止其不可逆聚集。被困在含 sHsps 聚集体中的底物可被 ATP 依赖性 Hsp70 和 Hsp100 伴侣蛋白有效地重折叠为天然结构。大多数 γ-变形菌都含有单个 sHsp（IbpA），而在肠杆菌目中的一部分细菌中，由于 ibpA 基因重复事件，已经进化出了一种由两个蛋白组成的 sHsp（IbpA 和 IbpB）系统。IbpA 和 IbpB 在功能上存在差异。纯化的 IbpA 可以稳定地与聚集的底物相互作用，但这两种 sHsps 都需要在底物变性步骤存在，以便随后进行有效的 Hsp70-Hsp100 依赖性底物重折叠。IbpA 和 IbpB 相互作用，影响彼此的表达水平和降解速率。然而，关于这两种 sHsps 如何相互作用以及正确发挥 sHsps 功能所需的基本构建块的关键信息尚不清楚。在这里，基于 NMR、质谱和交联研究，我们表明 IbpA-IbpB 异二聚体是肠杆菌目中两种 sHsp 系统的主要功能单元。异二聚体形成的原理类似于单个细菌 sHsps 同源二聚体描述的原理。IbpA 或 IbpB 晶状结构域的β5 和 β7 链形成的 β 发夹与异二聚体结构中另一个的 β-三明治结合。基于交联和分子动力学研究，我们还提出了两种 IbpA-IbpB 异二聚体在更高阶四聚体结构中的取向。
