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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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Comparison of the dimer interface surface structure of K182R, A183P ScMnSOD and K184R, L185P CaMnSODc to the WT proteins.The proteins are colored as: (A) WT ScMnSOD, green; (B) K182R, A183P ScMnSOD, red; (C) WT CaMnSODc, orange; (D) K184R, L185P CaMnSODc, blue. The dimer interfaces and hydrogen bonds are indicated as solid and dashed lines, respectively.
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pone-0062446-g003: Comparison of the dimer interface surface structure of K182R, A183P ScMnSOD and K184R, L185P CaMnSODc to the WT proteins.The proteins are colored as: (A) WT ScMnSOD, green; (B) K182R, A183P ScMnSOD, red; (C) WT CaMnSODc, orange; (D) K184R, L185P CaMnSODc, blue. The dimer interfaces and hydrogen bonds are indicated as solid and dashed lines, respectively.

Mentions: To investigate structural changes caused by the substitutions of Lys182 (Lys184) and Ala183 (Leu185) by arginine and proline, respectively, we solved the structures of the two RP-mutant proteins (Table 1). The tetrameric assemblies of WT and RP-mutant yeast MnSODs closely resemble each other (Figure S2). Superimpositions of all backbone atoms of the mutant subunit onto those of the WT subunit give root-mean-square deviations (RMSD) of 0.15 Å and 0.34 Å for ScMnSOD and CaMnSODc, respectively. The side chain of Arg182 (Arg184) of the mutants adopts a conformation different from that of Lys182 (Lys184) of the WT proteins (Figure 3). In two out of four chains in RP-mutant ScMnSOD, the chi2 and chi3 angles of the arginine shift by 5 and 127°, respectively (Figure 3A, 3B). In the other two chains, the chi1 angle of the arginine shifts by 132° (data not shown). These changes move Arg182 (Arg184) away from the dimer interface and thus open up a hole at the dimer interface of both RP-mutant ScMnSOD and RP-mutant CaMnSODc (Figure 3). The mutations also modify the hydrogen-bonding interactions surrounding residue 182 (184). Two hydrogen bonds, NZ(Lys184)···O(solv)···O(Ile129) and NZ(Lys184)···O(solv)···N(Gly131), are observed in WT CaMnSODc (Figure 3C), while in RP-mutant CaMnSODc the arginine is hydrogen bonded to Ile129 (Figure 3D).


Tetramerization reinforces the dimer interface of MnSOD.

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

Comparison of the dimer interface surface structure of K182R, A183P ScMnSOD and K184R, L185P CaMnSODc to the WT proteins.The proteins are colored as: (A) WT ScMnSOD, green; (B) K182R, A183P ScMnSOD, red; (C) WT CaMnSODc, orange; (D) K184R, L185P CaMnSODc, blue. The dimer interfaces and hydrogen bonds are indicated as solid and dashed lines, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646814&req=5

pone-0062446-g003: Comparison of the dimer interface surface structure of K182R, A183P ScMnSOD and K184R, L185P CaMnSODc to the WT proteins.The proteins are colored as: (A) WT ScMnSOD, green; (B) K182R, A183P ScMnSOD, red; (C) WT CaMnSODc, orange; (D) K184R, L185P CaMnSODc, blue. The dimer interfaces and hydrogen bonds are indicated as solid and dashed lines, respectively.
Mentions: To investigate structural changes caused by the substitutions of Lys182 (Lys184) and Ala183 (Leu185) by arginine and proline, respectively, we solved the structures of the two RP-mutant proteins (Table 1). The tetrameric assemblies of WT and RP-mutant yeast MnSODs closely resemble each other (Figure S2). Superimpositions of all backbone atoms of the mutant subunit onto those of the WT subunit give root-mean-square deviations (RMSD) of 0.15 Å and 0.34 Å for ScMnSOD and CaMnSODc, respectively. The side chain of Arg182 (Arg184) of the mutants adopts a conformation different from that of Lys182 (Lys184) of the WT proteins (Figure 3). In two out of four chains in RP-mutant ScMnSOD, the chi2 and chi3 angles of the arginine shift by 5 and 127°, respectively (Figure 3A, 3B). In the other two chains, the chi1 angle of the arginine shifts by 132° (data not shown). These changes move Arg182 (Arg184) away from the dimer interface and thus open up a hole at the dimer interface of both RP-mutant ScMnSOD and RP-mutant CaMnSODc (Figure 3). The mutations also modify the hydrogen-bonding interactions surrounding residue 182 (184). Two hydrogen bonds, NZ(Lys184)···O(solv)···O(Ile129) and NZ(Lys184)···O(solv)···N(Gly131), are observed in WT CaMnSODc (Figure 3C), while in RP-mutant CaMnSODc the arginine is hydrogen bonded to Ile129 (Figure 3D).

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