PMID:15100228
| Citation |
Tapley, TL and Vickery, LE (2004) Preferential substrate binding orientation by the molecular chaperone HscA. J. Biol. Chem. 279:28435-42 |
|---|---|
| Abstract |
HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99-103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations. |
| Links |
PubMed Online version:10.1074/jbc.M400803200 |
| Keywords |
Adenosine Diphosphate/chemistry; Adenosine Triphosphatases/chemistry; Adenosine Triphosphate/chemistry; Animals; Bicyclo Compounds/pharmacology; Cysteine/chemistry; Dose-Response Relationship, Drug; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/physiology; Fluorescent Dyes/pharmacology; HSP70 Heat-Shock Proteins/chemistry; HSP70 Heat-Shock Proteins/physiology; Iron-Sulfur Proteins/chemistry; Kinetics; Microscopy, Fluorescence; Models, Chemical; Models, Molecular; Molecular Chaperones/chemistry; Mutagenesis, Site-Directed; Mutation; Peptides/chemistry; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Rats; Spectrometry, Fluorescence; Tryptophan/chemistry |
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