Biological heterogeneity of the peptide-binding motif of the 70-kDa heat shock protein by surface plasmon resonance analysis.
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 B*2702-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 B*2702 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.
Maeda
Hideki
H
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.
Sahara
Hiroeki
H
Marine Biomedical Institute, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan. Electronic address: hsahara@sapmed.ac.jp.
Mori
Yoko
Y
Marine Biomedical Institute, Sapporo Medical University School of Medicine, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Torigo
Toshihiko
T
Departments of Pathology, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Kamiguchi
Kenjiro
K
Departments of Pathology, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Tamura
Yutaka
Y
Department of Bioinformatics Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chiba 260-8670, Japan.
Tamura
Yasuaki
Y
Departments of Pathology, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Hirata
Kouichi
K
Departments of Surgery, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
Sato
Noriyuki
N
Departments of Pathology, South 1 West 17, Chuo-ku, Sapporo 060-8556, Japan.
D000595:Amino Acid Sequence; D001665:Binding Sites; D015235:HLA-B Antigens / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D020349:Surface Plasmon Resonance
D015235:HLA-B Antigens; C042388:HLA-Bw4 antigen; D018840:HSP70 Heat-Shock Proteins