Interaction of the chaperone BiP with an antibody domain: implications for the chaperone cycle.
BiP is an Hsp70 homologue found in the endoplasmic reticulum of eukaryotic cells. Like other Hsp70 chaperones, BiP interacts with its substrate proteins in an ATP-dependent manner. The functional analysis has so far been performed mainly with short, synthetic peptides. Here, we present an experimental system that allows to study the partial reactions of the BiP chaperone cycle for a natural substrate protein domain in its soluble, stably unfolded conformation. This unfolded antibody domain forms a binary complex with BiP in the absence of ATP. The dissociation of the BiP dimer seems to be the rate-limiting step in this reaction. The BiP-C(H)3 complexes dissociate rapidly in the presence of ATP. The affinity for BiP-binding peptides and the non-native antibody domain was determined to be similar, suggesting that only the peptide binding site is involved in these interactions. Furthermore, these results imply that, also in the context of the antibody domain, an extended peptide sequence is recognized. However, the accessibility of the BiP-binding site in the non-native protein seems to influence the kinetics of complex formation.
Knarr
Gerhard
G
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.
Kies
Ursula
U
Bell
Stefan
S
Mayer
Marcus
M
Buchner
Johannes
J
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D002942:Circular Dichroism; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate