Biological Heterogeneity of the Peptide-binding Motif of the 70-kDa Heat Shock Protein by Surface Plasmon Resonance Analysis

Hideki Maeda,Hiroeki Sahara,Yoko Mori,Toshihiko Torigo,Kenjiro Kamiguchi,Yutaka Tamura,Yasuaki Tamura,Kouichi Hirata,Noriyuki Sato

doi:10.1074/jbc.m703436200

Abstract

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.

Highlights

Necessary to recognize binding sites of a target native protein, referred to as the binding motif
70-kDa heat shock protein family-binding motifs derived from observations of interactions with its many substrate proteins have been proposed, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance (SPR) analysis
We investigated the interaction of Hsp70 with the HLA-B*2702-derived peptide Bw4 (RENLRIALRY, HLA-B*2702 amino acids 75– 84) and its various truncated and substituted peptides using a Biacore system

Summary

Introduction

Necessary to recognize binding sites of a target native protein, referred to as the binding motif. It was reported that the host major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif interacted with a heptapeptide from a nucleoprotein of measles virus [11] and stimulated transcriptional activities of this virus [12, 13]. The 70-kDa heat shock protein family is capable of recognizing and binding to the structural region of a protein and to a specific localized peptide motif. 70-kDa heat shock protein family-binding motifs derived from observations of interactions with its many substrate proteins have been proposed, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance (SPR) analysis. The present study suggests a new binding motif between human Hsp and peptides derived from natural protein HLA-B*2702 and discloses characteristics of the Hsp70binding motif structure. Our findings may contribute to enhanced understanding of 70-kDa heat shock protein family binding determinants that may influence diverse cellular and physiological processes

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Conclusion