Tetramerization reinforces the dimer interface of MnSOD

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.

Figure 1. Alignment of MnSOD C-terminal Sequence.

Conserved residues and unconserved residues at dimer interface are highlighted in bold and shadowed in gray, respectively. The RP-mutations in ScMnSOD and CaMnSODc are highlighted in black.

Figure 2. The tetramer interfaces are highly disordered, when CaMnSODc is in the tetramer form.

The ribbon diagram of ScMnSOD (PDB code: 3LSU) is shown in Panel A. The four subunits are colored in: A, yellow; B, orange; C, green; D, cyan. The ribbon diagram of tetrameric CaMnSODc (PDB code: 3QVN) and the N-terminal helical region (residues 1–91) of a CaMnSODc monomer are shown in Panel B. The four subunits are colored in: A, yellow; B, orange; C, green; D, cyan. Manganese ions are indicated as purple spheres.

Figure 3. 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.

Figure 4. RP-mutant CaMnSODc is more subject to inactivation by pH than the wild type.

Rate constants as a function of pH were determined by fitting the disappearances of low doses of O₂ ⁻ ([O₂ ⁻]:[MnSOD] from 1–3) to first-order processes. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points circled and/or indicated with an arrow were measured after the pH was adjusted from 9–9.5 to neutral. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.

Figure 5. RP-mutant CaMnSODc is inactivated by heat like EcMnSOD.

Rate constants as a function of pH were determined by fitting the disappearances of low doses of O₂ ⁻ ([O₂ ⁻]:[MnSOD] from 1–3) to first-order processes. The enzymes were EcMnSOD (grey rectangle), WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The data points indicated with an arrow were obtained before the sample solution reached the desired temperature. All other data points were obtained after the sample solution was equilibrated to the desired temperature. The sample solutions contained 1 µM (in Mn) MnSOD in 10 mM potassium phosphate (pH 7), 10 mM sodium formate and 10 µM EDTA.

Figure 6. 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).

Figure 7. RP-mutant CaMnSODc is more subject to GdHCl-induced unfolding than the wild type.

The molar CD at 224 nm was used to monitor changes in α-helical structure content as a function of [GdHCl]. The enzymes were WT ScMnSOD (solid triangle), K182R, A183P ScMnSOD (hollow triangle), WT CaMnSODc (solid circle) and K184R, L185P CaMnSODc (hollow circle). The sample solutions contained 0.2 mg/mL (monomer concentration) MnSOD in 25 mM potassium phosphate (pH 7.4).

Figure 8. Thermostability of WT and RP-mutant ScMnSOD and CaMnSODc.

The S. cerevisiae enzymes in (A) are: (a) as-isolated ScMnSOD, (b) oxidized ScMnSOD and (c) as-isolated K182R, A183P ScMnSOD. The C. albicans enzymes in (B) are: (a) as-isolated CaMnSODc; (b) reduced CaMnSODc; (c) oxidized CaMnSODc and (d) as-isolated K184R, L185P CaMnSODc. Unfolding transitions are shown in black lines. The components (gray) were deconvoluted using a two-state irreversible model for WT ScMnSOD and a non-two-state reversible model for RP-mutant ScMnSOD, and WT and RP-mutant CaMnSODc. Reduced or oxidized enzymes were prepared by adding sodium hyposulfite or potassium permanganate to the sample solution prior to the DSC scan.