A ditryptophan cross-link is responsible for the covalent dimerization of humansuperoxide dismutase 1 during its bicarbonate-dependent peroxidase activity (original) (raw)
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Antioxidants & Redox Signaling, 2013
Aim: Human protein disulfide isomerase (hPDI) is a key enzyme and a redox-regulated chaperone responsible for oxidative protein folding in the endoplasmic reticulum. This work aims to reveal the molecular mechanism underlying the redox-regulated functions of hPDI by determining the crystal structures of hPDI in different redox states. Results: The structures of hPDI (abb¢xa¢) in both the reduced and oxidized states showed that the four thioredoxin domains of a, b, b¢, and a¢ are arranged as a horseshoe shape with two CGHC active sites, respectively, in domains a and a¢ facing each other at the two ends. In reduced hPDI, domains a, b, and b¢ line up in the same plane, whereas domain a¢ twists *45°out. The two active sites are 27.6 Å apart. In oxidized hPDI, the four domains are differently organized to stay in the same plane, and the distance between the active sites increases to 40.3 Å. In contrast to the closed conformation of reduced hPDI, oxidized hPDI exists in an open state with more exposed hydrophobic areas and a larger cleft with potential for substrate binding. Innovation: This is the first report of the high-resolution structures of hPDI containing all four domains in both the reduced and the oxidized states. It reveals the redox-regulated structural dynamic properties of the protein. Conclusion: The redox-regulated open/closed conformational switch of hPDI endows the protein with versatile target-binding capacities for its enzymatic and chaperone functions. Antioxid. Redox Signal. 19, 36-45.
Ditryptophan Cross-Links as Novel Products of Protein Oxidation
Journal of the Brazilian Chemical Society
Protein oxidation is an unavoidable consequence of aerobic metabolism. The oxidation of most proteins residues is non-repairable and may affect protein structure and function. In particular, protein cross-links arising from oxidative modifications are presumably toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain partially characterized. Up to very recently, ditryptophan cross-links (Trp-Trp), in particular, have been largely disregarded in the literature. Here, we briefly review studies showing that Trp-Trp cross-links can be formed in proteins exposed to a variety of oxidants. The challenges to fully characterize Trp-Trp cross-links are discussed as well as their potential roles in protein dysfunction and aggregation.
Oxidation-induced conformational change of Hsp33, monitored by NMR
Journal of the Korean Magnetic Resonance Society, 2015
Hsp33 is a prokaryotic molecular chaperon that exerts a holdase activity upon response to an oxidative stress at raised temperature. In particular, intramolecular disulfide bond formation between the four conserved cysteines that bind a zinc ion in reduced state is known to be critically associated with the redox sensing. Here we report the backbone NMR assignment results of the half-oxidized Hsp33, where only two of the four cysteines form an intramolecular disulfide bond. Almost all of the resolved peaks could be unambiguously assigned, although the total assignments extent reached just about 50%. Majority of the missing assignments could be attributed to a significant spectral collapse, largely due to the oxidation-induced unfolding of the C-terminal redox-switch domain. These results support two previous suggestions: conformational change in the first oxidation step is localized mainly in the C-terminal zinc-binding domain, and the half-oxidized form would be still inactive. However, some additional regions appeared to be potentially changed from the reduced state, which suggest that the half-oxidized conformation would be an intermediate state that is more labile to heat and/or further oxidation.
Journal of the Korean Magnetic Resonance Society, 2011
Hsp33, a prokaryotic molecular chaperone, exerts holdase activity in response to oxidative stress. In this study, the stepwise conformational change of Hsp33 upon oxidation was monitored by NMR. In order to overcome its high molecular weight (33 kDa as a monomer and 66 kDa as a dimer), spectra were simplified using a selectively [ 15 N]His-labeled protein. All of the eight histidines were observed in the TROSY spectrum of the reduced Hsp33. Among them, three peaks showed dramatic resonance shifts dependent on the stepwise oxidation, indicating a remarkable conformational change. The results suggest that unfolding of the linker domain is associated with dimerization, but not entire region of the linker domain is unfolded.
International Journal of Biological Macromolecules, 1998
The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) form. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature-or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 form oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.
Protein disulfide bond formation in the cytoplasm during oxidative stress
2004
The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both 4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.
Protein Science, 2008
Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a welldefined three-dimensional structure, which probably contains a-helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.
Per-deuteration and NMR experiments for the backbone assignment of 62 kDa protein, Hsp31
Journal of the Korean Magnetic Resonance Society, 2015
Hsp31 protein is one of the members of DJ-1 superfamily proteins and has a dimeric structure of which molecular weight (MW) is 62 kDa. The mutation of DJ-1 is closely related to early onset of Parkinson's disease. Hsp31 displays Zn +2-binding activity and was first reported to be a holding chaperone in E. coli. Its additional glyoxalase III active has recently been characterized. Moreover, an incubation at 60°C induces Hsp31 protein to form a high MW oligomer (HMW) in vitro, which accomplishes an elevated holding chaperone activity. The NMR technique is elegant method to probe any local or global structural change of a protein in responses to environmental stresses (heat, pH, and metal). Although the presence of the backbone chemical shifts (bbCSs) is a prerequisite for detailed NMR analyses of the structural changes, general HSQC-based triple resonance experiments could not be used for 62 kDa Hsp31 protein. Here, we prepared the per-deuterated Hsp31 and performed the TROSY-based triple resonance experiments for the bbCSs assignment. Here, detailed processes of per-deuteration and the NMR experiments are described for other similar NMR approaches.
Structure of Ero1p, Source of Disulfide Bonds for Oxidative Protein Folding in the Cell
Cell, 2004
FAD to catalyze disulfide bond formation and how disul-Weizmann Institute of Science fide bonds are relayed to proteins such as PDI are Rehovot 76100 poorly understood. Israel Insight into mechanisms of disulfide-forming enzymes 2 Department of Biology has come from studies of the Erv/ALR and quiescin/ Massachusetts Institute of Technology sulfhydryl oxidase (QSOX) protein families of FAD-Cambridge, Massachusetts 02139 dependent oxidases, which catalyze the following reaction: 2R Ϫ SH ϩ O 2 ↔ R Ϫ S Ϫ S Ϫ R ϩ H 2 O 2 (Hoober et al., 1996; Hoober and Thorpe, 1999). Erv/ALR modules, Summary which can be found as single-domain proteins or fused to thioredoxin-like domains in the QSOX enzymes (Cop-The flavoenzyme Ero1p produces disulfide bonds for pock et al., 1998), are found in a wide variety of proteins oxidative protein folding in the endoplasmic reticulum. targeted to different subcellular compartments and spe-Disulfides generated de novo within Ero1p are transcific oxidation reactions (Hoober et al., 1996; Becher et ferred to protein disulfide isomerase and then to subal., 1999; Senkevich et al., 2000; Gerber et al., 2001; strate proteins by dithiol-disulfide exchange reactions. Sevier and Kaiser, 2002). Erv2p, a member of this family, Despite this key role of Ero1p, little is known about resides in the yeast ER and generates a minor fraction the mechanism by which this enzyme catalyzes thiol of the disulfide bonds in that compartment (Sevier et al., oxidation. Here, we present the X-ray crystallographic 2001). Recently, we determined the three-dimensional structure of Ero1p, which reveals the molecular details structure of Erv2p by X-ray crystallography (Gross et of the catalytic center, the role of a CXXCXXC motif, al., 2002). The highly helical Erv2p represents a new and the spatial relationship between functionally sigclass of FAD binding fold and differs from the mixed nificant cysteines and the bound cofactor. Remark-␣/ structures of the thioredoxin-like oxidoreductases. ably, the Ero1p active site closely resembles that of Although the normal biological function of Erv2p in the the versatile thiol oxidase module of Erv2p, a protein ER is not known, overexpressed Erv2p can transfer diwith no sequence homology to Ero1p. Furthermore, sulfide bonds to PDI, presumably the reaction through both Ero1p and Erv2p display essential dicysteine mowhich high levels of Erv2p can bypass the need for tifs on mobile polypeptide segments, suggesting that Ero1p (Sevier et al . shuttling electrons to a rigid active site using a flexible
Archives of biochemistry and biophysics, 2014
Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase that has essential roles in redox protein folding. PDI has been associated with protective roles against protein aggregation, a hallmark of neurodegenerative diseases. Intriguingly, PDI has been detected in the protein inclusions found in the central nervous system of patients of neurodegenerative diseases. Oxidized proteins are also consistently detected in such patients, but the agents that promote these oxidations remain undefined. A potential trigger of protein oxidation is the bicarbonate-dependent peroxidase activity of the human enzyme superoxide dismutase 1 (hSOD1). Therefore, we examined the effects of this activity on PDI structure and activity. The results showed that PDI was oxidized to radicals that lead to PDI inactivation and aggregation. The aggregates are huge and apparently produced by covalent cross-links. Spin trapping experiments coupled with MS analysis indicated that at least 3 residues of...