Minami Y, Kawasaki H, Suzuki K, Yahara I
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
J Biol Chem. 1993 May 5;268(13):9604-10.
The mouse 90-kDa heat shock protein (HSP90) and Ca(2+)-calmodulin were cross-linked at an equimolar ratio using a carbodiimide zero-length cross-linker. To identify the calmodulin-binding domain(s) of HSP90, CNBr-cleaved peptide fragments of HSP90 were mixed with Ca(2+)-calmodulin and cross-linked. Amino acid sequence determination revealed that an HSP90 alpha-derived peptide starting at the 486th amino acid residue was contained in the cross-linked products, which contains a calmodulin-binding motif (from Lys500 to Ile520). A similar motif is present also in HSP90 beta (from Lys491 to Val511). The synthetic peptides corresponding to these putative calmodulin-binding sequences were found to be cross-linked with Ca(2+)-calmodulin and to prevent the cross-linking of HSP90 and Ca(2+)-calmodulin. Both HSP90 alpha and HSP90 beta bind Ca2+. The HSP90 peptides bind HSP90 and thereby inhibit the binding of Ca2+. In addition, the HSP90 peptides augment the self-oligomerization of HSP90 induced at elevated temperatures. These results suggest that the calmodulin-binding domain of HSP90 might interact with another part of the same molecule and that Ca(2+)-calmodulin might modulate the structure and function of HSP90 through abolishing the intramolecular interaction.
使用碳二亚胺零长度交联剂以等摩尔比将小鼠90 kDa热休克蛋白（HSP90）与Ca(2+)-钙调蛋白交联。为了鉴定HSP90的钙调蛋白结合结构域，将经CNBr裂解的HSP90肽片段与Ca(2+)-钙调蛋白混合并进行交联。氨基酸序列测定表明，交联产物中包含一个从第486个氨基酸残基开始的HSP90α衍生肽，该肽含有一个钙调蛋白结合基序（从Lys500到Ile520）。在HSP90β中也存在类似的基序（从Lys491到Val511）。发现与这些推定的钙调蛋白结合序列相对应的合成肽与Ca(2+)-钙调蛋白交联，并阻止HSP90与Ca(2+)-钙调蛋白的交联。HSP90α和HSP90β均结合Ca2+。HSP90肽结合HSP90，从而抑制Ca2+的结合。此外，HSP90肽增强了在高温下诱导的HSP90的自寡聚化。这些结果表明，HSP90的钙调蛋白结合结构域可能与同一分子的另一部分相互作用，并且Ca(2+)-钙调蛋白可能通过消除分子内相互作用来调节HSP90的结构和功能。
