Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• BiP/GRP78 is a crucial molecular chaperone involved in protein folding and quality control.
• Understanding the domain-specific functions of BiP/GRP78 is essential for elucidating its chaperone mechanism.

Purpose of the Study:

• To investigate the distinct roles of the N-terminal ATPase and C-terminal substrate-binding domains of BiP/GRP78.
• To determine how nucleotide and substrate binding influences BiP/GRP78 oligomerization and conformational states.

Main Methods:

• Engineered full-length recombinant BiP/GRP78 (FLAG-BiP.ent) with dual enterokinase cleavage sites.
• Investigated oligomerization dynamics, nucleotide binding effects, and domain dissociation using biochemical assays.
• Analyzed self-association of dissociated domains (N44.ent and C30.ent) under various conditions.

Main Results:

• FLAG-BiP.ent exists in a slow, concentration- and temperature-dependent equilibrium of multiple oligomeric species.
• ATP or AMP-PNP binding, along with peptidic substrate binding, induced depolymerization to monomeric BiP/GRP78.
• Cleavage revealed that the C-terminal substrate-binding domain (C30.ent) self-associates upon dissociation, while the N-terminal ATPase domain (N44.ent) remains monomeric.
• Peptide binding to C30.ent prevented its self-association, indicating domain-specific conformational changes.

Conclusions:

• Both N-terminal ATP binding and C-terminal peptide binding induce conformational changes within the C30 domain.
• These conformational changes are critical for the depolymerization of BiP/GRP78 from oligomeric to monomeric states.
• The study elucidates domain-specific contributions to BiP/GRP78's chaperone activity and regulation.