In Vitro Dissociation of BiP-Peptide Complexes Requires a Conformational Change in BiP after ATP Binding but Does Not Require ATP Hydrolysis
| Autor: | Linda M. Hendershot, James R. Gaut, Jueyang Wei |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Conformational change
genetic structures Protein Conformation Mutant Peptide macromolecular substances Plasma protein binding Biochemistry Adenosine Triphosphate Protein structure ATP hydrolysis Cricetinae Animals Point Mutation Nucleotide Binding site Endoplasmic Reticulum Chaperone BiP Molecular Biology Heat-Shock Proteins Adenosine Triphosphatases chemistry.chemical_classification Binding Sites Chemistry Cell Biology Recombinant Proteins Adenosine Diphosphate Kinetics Mutagenesis Site-Directed Carrier Proteins Molecular Chaperones Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 270:26677-26682 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.270.44.26677 |
| Popis: | In the present study, we produced single point mutations in the ATP binding site of hamster BiP, isolated recombinant proteins, and characterized them in terms of their affinity for ATP and ADP, their ability to undergo a conformational change upon nucleotide binding, and their rate of ATP hydrolysis. These analyses allowed us to classify the mutants into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins from BiP. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function. Addition of ATP to either wild-type BiP or the T229G mutant caused the in vitro release of bound peptide, confirming that ATP hydrolysis is not required for protein release. ATP did not dissociate G226D, G227D, or T37G mutant BiP-peptide complexes, suggesting that ATP binding to BiP is not sufficient for the release of bound peptides, but that an ATP-induced conformational change in BiP is necessary. The identification of BiP mutants that are defective in each of these steps of ATP hydrolysis will allow the in vivo dissection of the role of nucleotide in BiP's activity. |
| Databáze: | OpenAIRE |
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