Nemoto T K, Ono T, Kobayakawa T, Tanaka E, Baba T T, Tanaka K, Takagi T, Gotoh T
Department of Oral Biochemistry, Nagasaki University School of Dentistry, Nagasaki, Japan.
Eur J Biochem. 2001 Oct;268(20):5258-69. doi: 10.1046/j.0014-2956.2001.02457.x.
In the present study, we investigated the domain structure and domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7-Gly8, Arg336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90alpha. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1-Arg336; domain B, Glu337-Lys552; and domain C, Leu553-Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain-domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N-terminal two-thirds (subdomain BI) that interacted with domain A and the C-terminal one-third (subdomain BII) that interacted with domain C. The C-terminal two-thirds of domain A, i.e. Asp116-Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.
在本研究中，我们调查了真核生物HSP90的大肠杆菌同源物HtpG的结构域结构和结构域间相互作用。对重组HtpG进行有限蛋白酶解，发现了三个主要的胰蛋白酶切割位点，即Arg7-Gly8、Arg336-Glu337和Lys552-Leu553，其中后两个位点位于与人类HSP90α主要切割位点相当的位置。在非变性条件下用木瓜蛋白酶处理也得到了类似的模式，但在变性条件下则不然。因此，与HSP90一样，HtpG由三个结构域组成，即结构域A（Met1-Arg336）、结构域B（Glu337-Lys552）和结构域C（Leu553-Ser624）。表达了HtpG的各个结构域，并在非变性条件下通过聚丙烯酰胺凝胶电泳评估了它们之间的相互作用。结果揭示了两种结构域间相互作用：结构域B与同一多肽的结构域A相互作用，以及在二聚体中一个伙伴的结构域C与另一个伙伴的结构域B相互作用。结构域B在结构和功能上可分为两个亚结构域，与结构域A相互作用的N端三分之二（亚结构域BI）和与结构域C相互作用的C端三分之一（亚结构域BII）。结构域A的C端三分之二，即Asp116-Arg336，足以与结构域B结合。我们最终提出了HtpG二聚体的结构域组织方式。
