Identification of the N-terminal peptide binding site of glucose-regulated protein 94.
Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, we now locate this binding site by testing predictions of a molecular docking model. The best predicted site was on the opposite face of the beta sheet from the pan-HSP90 radicicol-binding pocket, in close proximity to a deep hydrophobic pocket. The peptide and radicicol-binding sites are distinct, as shown by the ability of a radicicol-refractive mutant to bind peptide. When the fluorophore acrylodan is attached to Cys117 within the hydrophobic pocket, its fluorescence is reduced upon peptide binding, consistent with proximity of the two ligands. Substitution of His125, which contacts the bound peptide, compromises peptide-binding activity. We conclude that peptide binds to the concave face of the beta sheet of the N-terminal domain, where binding is regulated during the action cycle of the chaperone.
Gidalevitz
Tali
T
Department of Pathology, the University of Chicago, Chicago, Illinois 60637, USA.
Biswas
Chhanda
C
Ding
Hua
H
Schneidman-Duhovny
Dina
D
Wolfson
Haim J
HJ
Stevens
Fred
F
Radford
Sheena
S
Argon
Yair
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C018551:glucose-regulated proteins