Identification of substrate binding site of GroEL minichaperone in solution.
It is difficult to obtain high-resolution structural information on the substrate-binding site of intact GroEL. But minichaperones, domains containing the peptide-binding site of GroEL, do constitute tractable systems for detailed studies. A peptide-binding site was located in crystals of a minichaperone and proposed to constitute a model for substrate-binding. We have now located the substrate binding site of the minichaperone GroEL(193-335) in solution by labelling it at various positions with a fluorescent probe and detecting which positions are perturbed on binding a denatured substrate. The fluorescence of a probe attached to a cysteine residue engineered at position 228 (N terminus of helix H8), 241 (helix H8), 261 (helix H9), or 267 (helix H9) was affected significantly by binding of substrate. But there was little change for a label at positions 193, 212, 217 or 293. The dissociation constants between substrates and minichaperone were evaluated from fluorescence anisotropy assays. The effects of salt and temperature were the same as those with intact GroEL. These results indicate that the region around helices H8 and H9 is the substrate-binding site for the apical domain fragment. Intriguingly, the same site is involved in the binding of GroES. Thus, an important function of GroES in the regulation of the activity of GroEL for substrates is to displace the bound substrate by competing for its binding site.
Tanaka
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Department of Polymer Science and Engineering, Kyoto Institute of Technology, Sakyo, Kyoto, 606-8585, Japan.
Fersht
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AR
D001665:Binding Sites; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry*; D004926:Escherichia coli; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007768:Lactalbumin / Q000737:chemistry; D008958:Models, Molecular; D010434:Pepsin A / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
D018835:Chaperonin 10; D018834:Chaperonin 60; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D007768:Lactalbumin; D010434:Pepsin A