Preferential substrate binding orientation by the molecular chaperone HscA.
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.
Tapley
Tim L
TL
Department of Physiology and Biophysics, University of California-Irvine, Irvine, CA 92697, USA.
Vickery
Larry E
LE
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D001643:Bridged Bicyclo Compounds; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D007506:Iron-Sulfur Proteins; C411665:IscU protein, E coli; D018832:Molecular Chaperones; D010455:Peptides; D020033:Protein Isoforms; C085650:hscA protein, E coli; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; D003545:Cysteine; C028381:monobromobimane