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Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

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RP-mutant CaMnSODc is susceptible to dimer dissociation.HPLC-SEC profiles of WT (solid line) and K182R, A183P (dashed line) ScMnSOD are shown in Panel A. Inset: The plot of the molecular weight of the five standards (square), ScMnSOD tetramer (circle) and CaMnSODc dimer (triangle down) and monomer (triangle up) versus their retention time. The column was calibrated using five standards: 1) bovine thyroglobulin (670 kDa), 2) bovine γ-globulin (158 kDa), 3) ovalbumin (44 kDa), 4) horse myoglobin (17 kDa), and 5) Vitamin B12 (1.35 kDa). HPLC-SEC profiles of WT (solid line) and K184R, L185P (dashed line) CaMnSODc are shown in Panel B. Deconvoluted peaks are shown in grey lines. The protein concentration relative to monomer was 1 µM (a), 750 nM (b), 500 nM (c) and 200 nM (d). The elution buffer contained 10 mM potassium phosphate (pH 6.7).
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pone-0062446-g006: RP-mutant CaMnSODc is susceptible to dimer dissociation.HPLC-SEC profiles of WT (solid line) and K182R, A183P (dashed line) ScMnSOD are shown in Panel A. Inset: The plot of the molecular weight of the five standards (square), ScMnSOD tetramer (circle) and CaMnSODc dimer (triangle down) and monomer (triangle up) versus their retention time. The column was calibrated using five standards: 1) bovine thyroglobulin (670 kDa), 2) bovine γ-globulin (158 kDa), 3) ovalbumin (44 kDa), 4) horse myoglobin (17 kDa), and 5) Vitamin B12 (1.35 kDa). HPLC-SEC profiles of WT (solid line) and K184R, L185P (dashed line) CaMnSODc are shown in Panel B. Deconvoluted peaks are shown in grey lines. The protein concentration relative to monomer was 1 µM (a), 750 nM (b), 500 nM (c) and 200 nM (d). The elution buffer contained 10 mM potassium phosphate (pH 6.7).

Mentions: The oligomeric states of the as-isolated proteins were investigated by HPLC-SEC. When the protein concentration with respect to monomer was varied from 10 µM to 200 nM, WT and RP-mutant ScMnSOD both eluted solely as tetramers (Figure 6A), and WT CaMnSODc eluted solely as dimers (Figure 6B). The different oligomeric states of WT ScMnSOD and WT CaMnSODc are unlikely to result from differences in metallation states (Table S1), since WT ScMnSOD and WT CaMnSODc, when both metallated with ∼0.6 Mn per monomer, elute as tetramers and dimers, respectively [9].


Tetramerization reinforces the dimer interface of MnSOD.

Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS - PLoS ONE (2013)

RP-mutant CaMnSODc is susceptible to dimer dissociation.HPLC-SEC profiles of WT (solid line) and K182R, A183P (dashed line) ScMnSOD are shown in Panel A. Inset: The plot of the molecular weight of the five standards (square), ScMnSOD tetramer (circle) and CaMnSODc dimer (triangle down) and monomer (triangle up) versus their retention time. The column was calibrated using five standards: 1) bovine thyroglobulin (670 kDa), 2) bovine γ-globulin (158 kDa), 3) ovalbumin (44 kDa), 4) horse myoglobin (17 kDa), and 5) Vitamin B12 (1.35 kDa). HPLC-SEC profiles of WT (solid line) and K184R, L185P (dashed line) CaMnSODc are shown in Panel B. Deconvoluted peaks are shown in grey lines. The protein concentration relative to monomer was 1 µM (a), 750 nM (b), 500 nM (c) and 200 nM (d). The elution buffer contained 10 mM potassium phosphate (pH 6.7).
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pone-0062446-g006: RP-mutant CaMnSODc is susceptible to dimer dissociation.HPLC-SEC profiles of WT (solid line) and K182R, A183P (dashed line) ScMnSOD are shown in Panel A. Inset: The plot of the molecular weight of the five standards (square), ScMnSOD tetramer (circle) and CaMnSODc dimer (triangle down) and monomer (triangle up) versus their retention time. The column was calibrated using five standards: 1) bovine thyroglobulin (670 kDa), 2) bovine γ-globulin (158 kDa), 3) ovalbumin (44 kDa), 4) horse myoglobin (17 kDa), and 5) Vitamin B12 (1.35 kDa). HPLC-SEC profiles of WT (solid line) and K184R, L185P (dashed line) CaMnSODc are shown in Panel B. Deconvoluted peaks are shown in grey lines. The protein concentration relative to monomer was 1 µM (a), 750 nM (b), 500 nM (c) and 200 nM (d). The elution buffer contained 10 mM potassium phosphate (pH 6.7).
Mentions: The oligomeric states of the as-isolated proteins were investigated by HPLC-SEC. When the protein concentration with respect to monomer was varied from 10 µM to 200 nM, WT and RP-mutant ScMnSOD both eluted solely as tetramers (Figure 6A), and WT CaMnSODc eluted solely as dimers (Figure 6B). The different oligomeric states of WT ScMnSOD and WT CaMnSODc are unlikely to result from differences in metallation states (Table S1), since WT ScMnSOD and WT CaMnSODc, when both metallated with ∼0.6 Mn per monomer, elute as tetramers and dimers, respectively [9].

Bottom Line: Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure.Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD.We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or "loose tetramer" in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.

Show MeSH
Related in: MedlinePlus