Schleiff E, Turnbull J L
Department of Biochemistry, McGill University, Montreal, Canada.
Biochemistry. 1998 Sep 22;37(38):13052-8. doi: 10.1021/bi980746y.
hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.
hTom20是一种参与蛋白质转运的线粒体外膜受体。该蛋白的胞质结构域（氨基酸30 - 145）及其选定的截短版本被过表达并纯化，以研究其结构 - 功能关系。我们的研究表明，胞质结构域的二级结构对盐酸胍和尿素的变性具有很强的抗性，主要通过疏水相互作用得以稳定。然而，N端靶向信号结合结构域（氨基酸30 - 90）的三级结构则更具灵活性。胞质结构域的前30个氨基酸（氨基酸30 - 60）参与识别N端靶向信号，并在脂质表面稳定胞质结构域。此外，我们发现，具体而言，氨基酸30 - 48与膜表面相互作用；包含氨基酸48 - 145的构建体仅在存在N端靶向信号肽的情况下才会结合到膜表面。hTom20的C端区域（氨基酸141 - 145）与hTom20的N端区域相互作用，有助于稳定N端靶向信号结合结构域的正确构象。最后，hTom20与融合了二氢叶酸还原酶的鸟氨酸氨甲酰基转移酶前体的N端靶向信号的相互作用非常弱（解离常数Kd = 8微摩尔），如果这种相互作用是由导入受体复合物精心安排的一系列作用中的第一步，以将靶向蛋白导入线粒体，那么这是可以预期的。
