Department of Biochemistry, University of Venda, Private Bag X5050, Thohoyandou 0950, South Africa.
Structural Biology Research Unit, Department of Integrative Biomedical Sciences, University of Cape Town, Observatory 7925, South Africa.
Int J Mol Sci. 2021 Feb 23;22(4):2226. doi: 10.3390/ijms22042226.
Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.
寄生虫，特别是顶复门的寄生虫，含有一种胞质定位的经典 Hsp70 伴侣蛋白。这种蛋白质的一个显著特征是存在 GGMP 重复残基，夹在 α-螺旋盖和 C 末端 EEVD 基序之间。Hsp70 中 GGMP 重复的作用仍然未知。在本研究中，我们在 （PfHsp70-1）和嵌合蛋白（KPf）中引入了胞质定位的 Hsp70-1 的 GGMP 突变，该嵌合蛋白由 DnaK 的 ATP 酶结构域与 PfHsp70-1 的 C 末端底物结合结构域融合而成。使用 细胞进行的互补测定表明，GGMP 基序对于嵌合蛋白 KPf 的伴侣蛋白功能是必需的。有趣的是，将 PfHsp70-1 的 GGMP 基序插入 DnaK 会导致在暴露于升高的生长温度的 细胞中出现致死表型。通过生化和生物物理测定，我们确定 GGMP 基序解释了 PfHsp70-1 的基础 ATP 酶活性升高。此外，我们证明该基序对于伴侣蛋白与肽底物和共伴侣蛋白 PfHop 的相互作用很重要。我们的研究结果表明，GGMP 可能解释了 PfHsp70-1 的特殊伴侣蛋白功能和据称的高催化效率。
