title: Structural plasticity and noncovalent substrate binding in the GroEL apical domain. A study using electrospay ionization mass spectrometry and fluorescence binding studies.
abstract: Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.
authors.affiliation: Astbury Centre for Structural Molecular Biology & School of Biochemistry & Molecular Biology, The University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Achim
authors.initials: A
authors.lastname: Brinker
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Coyle
authors.forename: Frank
authors.initials: F
authors.lastname: Weber
authors.forename: Markus
authors.initials: M
authors.lastname: Kaiser
authors.forename: Luis
authors.initials: L
authors.lastname: Moroder
authors.forename: Mark R
authors.initials: MR
authors.lastname: Parsons
authors.forename: Joachim
authors.initials: J
authors.lastname: Jager
authors.forename: Ulrich F
authors.initials: UF
authors.lastname: Hartl
authors.forename: Manajit
authors.initials: M
authors.lastname: Hayer-Hartl
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D001665:Binding Sites; D018834:Chaperonin 60 / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D018834:Chaperonin 60; D007477:Ions; D008024:Ligands