Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP.
To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.
Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60607-7173, USA.
M
M
Chevalier
L
L
King
C
C
Wang
M J
MJ
Gething
E
E
Elguindi
S Y
SY
Blond
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D002352:Carrier Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D004765:Enteropeptidase / Q000378:metabolism; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D001704:Biopolymers; D002352:Carrier Proteins; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D004765:Enteropeptidase