Maeda Hideki, Sahara Hiroeki, Mori Yoko, Torigo Toshihiko, Kamiguchi Kenjiro, Tamura Yutaka, Tamura Yasuaki, Hirata Kouichi, Sato Noriyuki
Departments of Surgery, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan; Departments of Pathology, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Marine Biomedical Institute, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
J Biol Chem. 2007 Sep 14;282(37):26956-26962. doi: 10.1074/jbc.M703436200. Epub 2007 Jul 11.
70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.
70 kDa热休克蛋白家族是一种分子伴侣，可在细胞质中与多种客户蛋白和肽结合。多项研究通过噬菌体展示文库筛选等传统技术揭示了70 kDa热休克蛋白家族与细胞质蛋白之间的结合基序。然而，基于表面等离子体共振分析确定的动力学参数的结合基序却鲜为人知。我们使用基于表面等离子体共振分析的Biacore系统，研究了人白细胞抗原B2702衍生肽Bw4（RENLRIALRY）与主要可诱导的胞质70 kDa热休克蛋白（Hsp70）的结合基序。Hsp70与Bw4相互作用的解离常数（K(D)）值为1.8×10^(-6) m。对截短的Bw4变体肽的分析表明，Hsp70的结合基序为七个残基，即LRIALRY。为了进一步研究该基序的特征，合成了126个源自Bw4的肽，每个肽都有一个单氨基酸取代，并分析了它们与Hsp70的结合亲和力。有趣的是，当任何位置的残基被酸性（天冬氨酸和谷氨酸）残基取代时，Hsp70的结合亲和力就会丧失。相反，如果取代残基是芳香族（色氨酸、酪氨酸和苯丙氨酸）或任何位置的精氨酸残基，则Hsp70的结合亲和力得以维持。因此，本研究提出了Hsp70与源自天然蛋白人白细胞抗原B2702的肽之间的新结合基序，也可能阐明Hsp70结合特性的一些特征，增进我们对可能影响多种细胞和生理过程的Hsp70结合决定因素的理解。
