An unexpected second binding site for polypeptide substrates is essential for Hsp70 chaperone activity

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Science

Background:

• Heat shock proteins of 70 kDa (Hsp70s) are crucial molecular chaperones involved in protein folding and homeostasis.
• Existing structural data indicated a single substrate-binding site within the Hsp70 substrate-binding domain (SBD).
• This single binding site posed challenges in explaining the high efficiency of Hsp70 chaperone functions.

Purpose of the Study:

• To investigate the mechanism behind the efficient chaperone activity of Hsp70s.
• To identify potential additional binding sites within Hsp70s.
• To elucidate the role of any newly discovered binding sites in Hsp70 function.

Main Methods:

• Purified Hsp70 proteins were utilized.
• Site-directed mutagenesis was employed to probe protein function.
• Biochemical assays, including fluorescence polarization and luciferase-refolding assays, were performed.
• Cross-linking studies were conducted to map binding site proximity.

Main Results:

• A previously unrecognized second peptide-binding site (P2) was discovered in Hsp70s.
• Biochemical analyses confirmed the essential role of this P2 site in Hsp70 chaperone activity.
• Mutagenesis and cross-linking data indicated the P2 site is located in the SBD, adjacent to the primary binding site.
• The two binding sites were suggested to cooperate in the protein folding process.

Conclusions:

• Hsp70s possess two cooperating peptide-binding sites within their SBD.
• The newly identified P2 site is critical for Hsp70s' ability to facilitate protein folding.
• This discovery provides a revised mechanistic understanding of Hsp70 chaperone function.