The Crystal Structure of the Reduced, Zn2+-Bound Form of the B. subtilis Hsp33 Chaperone and Its Implications for the Activation Mechanism

AbstractThe bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress. In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal. This leads to the formation of the biologically active Hsp33 dimer. The crystal structure of the N-terminal domain of the E. coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known. Here we report the crystal structure of the full-length B. subtilis Hsp33 in the reduced form. The structure of the N-terminal, dimerization domain is similar to that of the E. coli protein, although there is no domain swapping. The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates. We propose a structure-based activation pathway for Hsp33

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the key redox-sensitive proteins whose activity is largely affected by oxidative modifications at its highly reactive cysteine residue in the enzyme's active site (Cys149). Prolonged exposure to oxidative stress may cause, inter alia, the formation of intermolecular disulfide bonds leading to accumulation of GAPDH aggregates and ultimately to cell death. Recently these anomalies have been linked with the pathogenesis of Alzheimer's disease. Novel evidences indicate that low molecular compounds may be effective inhibitors potentially preventing the GAPDH translocation to the nucleus, and inhibiting or slowing down its aggregation and oligomerization. Therefore, we decided to establish the ability of naturally occurring compound, piceatannol, to interact with GAPDH and to reveal its effect on functional properties and selected parameters of the dehydrogenase structure. The obtained data revealed that piceatannol binds to GAPDH. The ITC analysis indicated that one molecule of the tetrameric enzyme may bind up to 8 molecules of polyphenol (7.3 ± 0.9). Potential binding sites of piceatannol to the GAPDH molecule were analyzed using the Ligand Fit algorithm. Conducted analysis detected 11 ligand binding positions. We indicated that piceatannol decreases GAPDH activity. Detailed analysis allowed us to presume that this effect is due to piceatannol ability to assemble a covalent binding with nucleophilic cysteine residue (Cys149) which is directly involved in the catalytic reaction. Consequently, our studies strongly indicate that piceatannol would be an exceptional inhibitor thanks to its ability to break the aforementioned pathologic disulfide linkage, and therefore to inhibit GAPDH aggregation. We demonstrated that by binding with GAPDH piceatannol blocks cysteine residue and counteracts its oxidative modifications, that induce oligomerization and GAPDH aggregation

Proposed mechanism for the conjugation of catechol moiety of piceatannol to a nucleophilic thiol group (Cys149) in GAPDH molecule.

<p>Proposed mechanism for the conjugation of catechol moiety of piceatannol to a nucleophilic thiol group (Cys149) in GAPDH molecule.</p

Binding parameters of the interaction of GAPDH protein with piceatannol in water solution at 25°C determined by isothermal titration calorimetry.

<p>Binding parameters of the interaction of GAPDH protein with piceatannol in water solution at 25°C determined by isothermal titration calorimetry.</p

Most frequently interactions of piceatannol with amino acid residues (the catalytic region of enzyme) active site of the O subunit of GAPDH.

<p>Most frequently interactions of piceatannol with amino acid residues (the catalytic region of enzyme) active site of the O subunit of GAPDH.</p

The intensity of thioflavin-T binding-dependent fluorescence of native GAPDH, GAPDH treated with piceatannol (50 μM), GAPDH treated with hydrogen peroxide (3 mM) and GAPDH preincubated for 30 minutes with piceatannol or resveratrol (50 μM) and afterwards treated with H₂O₂ at 37°C for the indicated times is shown.

<p>Data are means ± SD of n = 4–8 independent measurements.</p

Functional consequences of piceatannol binding to glyceraldehyde-3-phosphate dehydrogenase - Fig 9

<p>The micrographs of <b>(A)</b> GAPDH, <b>(B)</b> GAPDH and H₂O₂, <b>(C)</b> GAPDH with piceatannol and H₂O₂, <b>(D)</b> GAPDH with resveratrol and H₂O₂, from light microscope and transmission electron microscope. Bright field—left panel, dark field—middle panel and electron micrographs-right panel.</p

Far-UV CD spectra of native GAPDH and GAPDH treated with 32 μM of piceatannol at room temperature for 50 and 100 min of incubation.

<p>The data are expressed as molar residue ellipticity.</p

Most frequently occurring interactions of piceatannol at the active site of the O subunit of GAPDH.

<p>Number in parentheses represents how many times the residue was involved in interaction/30 best poses.</p