Radicicol-sensitive peptide binding to the N-terminal portion of GRP94.
GRP94 is a molecular chaperone that carries immunologically relevant peptides from cell to cell, transferring them to major histocompatibility proteins for presentation to T cells. Here we examine the binding of several peptides to recombinant GRP94 and study the regulation and site of peptide binding. We show that GRP94 contains a peptide-binding site in its N-terminal 355 amino acids. A number of peptides bind to this site with low on- and off-rates and with specificity that is distinct from that of another endoplasmic reticulum chaperone, BiP/GRP78. Binding to the N-terminal fragment is sufficient to account for the peptide binding activity of the entire molecule. Peptide binding is inhibited by radicicol, a known inhibitor of the chaperone activities of HSP90-family proteins. However, the peptide-binding site is distinct from the radicicol-binding pocket, because both can bind to the N-terminal fragment simultaneously. Furthermore, peptide binding does not cause the same conformational change as does binding of radicicol. When the latter binds to the N-terminal domain, it induces a conformational change in the downstream, acidic domain of GRP94, as measured by altered gel mobility and loss of an antibody epitope. These results relate the peptide-binding activity of GRP94 to its other function as a chaperone.
Department of Pathology, Committees on Cell Physiology and Immunology, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.
Shawn
S
Vogen
Tali
T
Gidalevitz
Chhanda
C
Biswas
Birgitte B
BB
Simen
Eytan
E
Stein
Funda
F
Gulmen
Yair
Y
Argon
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology*; D008024:Ligands; D018942:Macrolides; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D007783:Lactones; D008024:Ligands; D018942:Macrolides; D008565:Membrane Proteins; D010455:Peptides; D011994:Recombinant Proteins; C018551:glucose-regulated proteins; C035359:monorden