Nemoto T, Matsusaka T, Ota M, Takagi T, Collinge D B, Walther-Larsen H
Department of Biochemistry, Iwate Medical University School of Dentistry.
J Biochem. 1996 Aug;120(2):249-56. doi: 10.1093/oxfordjournals.jbchem.a021406.
The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 (HSP90 alpha) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90 alpha were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90 alpha. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90 alpha and the C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.
94 kDa葡萄糖调节蛋白（GRP94）是90 kDa热休克蛋白（HSP90）家族的成员。在本研究中，我们在大肠杆菌中表达了大麦（Hordeum vulgare L.）GRP94和人HSP90的α异构体（HSP90α），并比较了它们形成二聚体的能力。天然聚丙烯酰胺凝胶电泳显示GRP94（氨基酸69 - 809）和HSP90α的全长形式以二聚体状态存在。GRP94的C末端326个氨基酸或HSP90α的C末端200个氨基酸足以形成二聚体。用凝血酶对GRP94 C末端一半进行有限蛋白酶解，得到一个16 kDa的片段，该片段是通过切割Arg710 - His711或Arg735 - Leu736键从GRP94的C末端产生的。这些切割位点即使不完全相同，也几乎等同于HSP90α的蛋白酶解区域。它们的结构相似性促使我们利用共表达系统研究这两种蛋白质形成异源二聚体复合物的可能性。一种仅特异性捕获该复合物的两步亲和层析显示，HSP90α的C末端200个氨基酸和GRP94的C末端326个氨基酸分别与HSP90α和GRP94相互作用。然而，GRP94的C末端326个氨基酸未能与HSP90α形成复合物。总之，这些结果表明这两种HSP90家族成员蛋白的一般二聚体构象具有相似性，但表明这种相似性不足以允许形成异源二聚体。
