Mass Spectrometry Unravels Disulfide Bond Formation as the Mechanism That Activates a Molecular Chaperone

Abstract

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.Hsp33 is a newly discovered heat shock protein that functions as a highly efficient molecular chaperone (1). Hsp33 protects bacterial cells from deleterious effects caused by oxidants like H 2 O 2 showing that it plays a role in the bacterial defense system against oxidative stress. Hsp33 is distinguished from all other known chaperone proteins by the finding that Hsp33 is functionally regulated at posttranslational level, by the redox conditions of the environment (1) (reviewed in Refs. 2 and 3). Elevated H 2 O 2 concentrations induce the chaperone functions of Hsp33 (1), but the precise molecular mechanism that translates the changes of the cellular redox environment into differences in chaperone activity is not yet understood.The Hsp33 amino acid sequence contains a novel conserved motif consisting of four cysteinyl residues near the C terminus of the protein. These cysteinyl residues form a C-X-C motif and a C-Y-Z-C motif (Fig. 1), separated by 27-30 amino acid residues in Escherichia coli Hsp33 and its homologues. These four cysteinyl residues are present in all 27 known Hsp33 homologues suggesting that they play an important role in the function of Hsp33. In addition, two further cysteinyl residues, Cys 141 and Cys 239 , are present in Hsp33. Cys 239 is very poorly conserved occurring in only 4 of the 27 known Hsp33 homologues. Cys 141 is moderately conserved and is present in 10 of the 27 Hsp33 homologues known so far. We have postulated that disulfide bond formation is involved in the activation process of Hsp33. Thus, analysis of the disulfide bond status and connectivities in inactive and in active Hsp33 seemed very important to further understand the regulation of this efficient molecular chaperone.Two complementary mass spectrometry-based strategies are suitable for determining the presence and locations of disulfide bonds in proteins. Which one is superior is dependent on the proximity of the involved cysteinyl residues in the amino acid sequence. Both approaches involve cleavage of the protein by enzymatic or chemical means under conditions that minimize disulfide bond scrambling (4 -6). The first approach can be used when the proteolytically derived peptides contain zero or one cysteinyl residue. The peptide mixtures are directly analyzed by mass spectrometric peptide mapping. Molecular mass analyses identify the peptides and disulfide-bonded dipeptides by assigning the observed ion signals to the corresponding calculat...

The redox-switch domain of Hsp33 functions as dual stress sensor

“…This has been observed for other thiol-containing peptides and agrees with our original mass spectrometry studies, where only a very small signal for the disulfide-bonded 232-236 peptide was detected 11 . Nevertheless, our identification of an oxidation intermediate shows that the oxidative thiol modifications occurring at 30 °C differ from those at 43 °C and suggests formation of an inactive oxidation intermediate at 30 °C.…”

“…The presence of this zinc center apparently keeps Hsp33 monomeric and functionally inactive. Upon exposure of Hsp33 to oxidative stress, the nearby cysteines form intramolecular disulfide bonds, inducing zinc release 7,11 . Large conformational rearrangements and partial unfolding then lead to the dimerization of Hsp33, a crucial step that fully activates Hsp33's chaperone function 7,12 .…”

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…Reduced Hsp33 is monomeric and inactive, whereas the oxidized chaperone is dimeric and functional (156a, 344a). The activity switch is brought about by the reversible disulfide bond formation between two pairs of cysteines that coordinate zinc in the reduced state (14). Zinc release and concomitant dimerization of Hsp33 induces structural rearrangements that lead to the formation of potential binding sites for unfolded proteins.…”

Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding

“…The four absolutely conserved cysteines that constitute this redox switch are kept in the reduced deprotonated thiolate anion state and together coordinate one zinc(II) ion (K D ϳ 10 Ϫ18 M) (3). Under oxidative stress conditions, these four cysteines release zinc and rapidly form two intramolecular disulfide bonds, connecting the two pairs of neighboring cysteines, Cys 232 with Cys 234 and Cys 265 with Cys 268 (4). Disulfide bond formation and concomitant zinc release then induces the dimerization of two oxidized Hsp33 monomers (5).…”