Activation of the redox-regulated chaperone Hsp33 by domain unfolding.
The molecular chaperone Hsp33 in Escherichia coli responds to oxidative stress conditions with the rapid activation of its chaperone function. On its activation pathway, Hsp33 progresses through three major conformations, starting as a reduced, zinc-bound inactive monomer, proceeding through an oxidized zinc-free monomer, and ending as a fully active oxidized dimer. While it is known that Hsp33 senses oxidative stress through its C-terminal four-cysteine zinc center, the nature of the conformational changes in Hsp33 that must take place to accommodate this activation process is largely unknown. To investigate these conformational rearrangements, we constructed constitutively monomeric Hsp33 variants as well as fragments consisting of the redox regulatory C-terminal domain of Hsp33. These proteins were studied by a combination of biochemical and NMR spectroscopic techniques. We found that in the reduced, monomeric conformation, zinc binding stabilizes the C terminus of Hsp33 in a highly compact, alpha-helical structure. This appears to conceal both the substrate-binding site as well as the dimerization interface. Zinc release without formation of the two native disulfide bonds causes the partial unfolding of the C terminus of Hsp33. This is sufficient to unmask the substrate-binding site, but not the dimerization interface, rendering reduced zinc-free Hsp33 partially active yet monomeric. Critical for the dimerization is disulfide bond formation, which causes the further unfolding of the C terminus of Hsp3 and allows the association of two oxidized Hsp33 monomers. This then leads to the formation of active Hsp33 dimers, which are capable of protecting cells against the severe consequences of oxidative heat stress.
Graf
Paul C F
PC
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048, USA.
Martinez-Yamout
Maria
M
VanHaerents
Stephen
S
Lilie
Hauke
H
Dyson
H Jane
HJ
Jakob
Ursula
U
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014461:Ultracentrifugation; D015032:Zinc / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C492165:HSP33 protein, E coli; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D006861:Hydrogen Peroxide; D015032:Zinc; D003545:Cysteine; D010100:Oxygen