Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase

doi:10.1016/0006-291X(89)92486-8

Biochemical and Biophysical Research Communications

Volume 160, Issue 2, 28 April 1989, Pages 677-681

https://doi.org/10.1016/0006-291X(89)92486-8 Get rights and content

Abstract

The Cu, Zn superoxide dismutase from yeast was crystallized in the orthorhombic space group P21212 with unit cell dimension a=105.1A˚, b=142.2A˚, c=62.1A˚. The crystals grow in 25 mM citrate, 10 mM phosphate buffer pH 6.5, and 6% (W/V) polyethylene glycol, with a V_m of 3,4A˚³ /dalton, for two dimers/asymmetric unit. The crystals were unstable in the mother liquor, but were stabilized by transfer to a 35% polyethylene glycol solution. This crystalline form diffracts at high resolution and is suitable for determination of the atomic structure. The three dimensional structure of the yeast enzyme could be model-built by computer graphics techniques using the bovine enzyme atomic coordinates as template. The proposed model requires removal of some salt bridges and non equivalence of the metal-binding sites in the subunits, in line with reported functional properties of the yeast enzyme.

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Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans
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Citation Excerpt :
SEQBOOT, PROTDIST, FITCH, and CONSENSUS programs were all included in the PHYLIP package, version 3.573c (Felsenstein, 1993). Three-dimensional structures of CnSOD1s were computer modeled using the S. cerevisiae SOD1 crystal structure as a template (Frigerio et al., 1989). The amino acid sequence of CnSOD1 was compared with the S. cerevisiae sequence using the NORMPAIRS and MULT-TREE programs in the multiple sequence comparison package MULTALIGN (Barton and Sternberg, 1987).
Cryptococcus neoformans (Cn), causal agent of fungal meningoencephalitis, has three varieties with variable host predilection. To explore mechanisms for these pathogenic differences, we have characterized Cu,Zn SOD gene (CnSOD1). A Saccharomyces cerevisiae sod1Δ mutant was complemented with Cn var. grubii yeast expression library. The complementing clone had an ORF of 462 bp and the deduced 154 aa sequence showed 61% identity with S. cerevisiae SOD1 and 53–65% with other eukaryotic SOD1s. Cn var. grubii CnSOD1 cDNA was used to clone corresponding cDNAs from var. neoformans and var. gattii. ORFs from three varieties revealed 20–29% differences in deduced aa (s) with a significant 6% non-synonymous aa substitution between Cn var. grubii and Cn var. gattii. Cosmid library screening and PCR cloning were used to obtain genomic SOD1, which was split by five introns with identical placements and a typical 5′ splice junction sequence, GTNNGY. These introns also showed a large nt variation among the three Cn varieties. Phylogenetic analyses revealed CnSOD1 to be in a group distinct from other eukaryotic SOD1s and with a significant divergence of the var. grubii from var. gattii. The CnSOD1 -deduced protein was modeled based on the crystal structure of S. cerevisiae SOD1, which showed an excellent fit. Most of the non-synonymous aa substitutions occurred on the outside of the molecule and these may contribute to differences in antigenicity among the three varieties. Notably, Cn var. neoformans and var. gattii Cu,Zn SOD had three substitutions of glycine (Gly26, Gly92 and Gly123 for Asn26, Ser92 and Ser123) that may contribute to the observed lower thermostability of this enzyme vis-a-vis Cn var. grubii. This is the first nucleotide and structural comparison of a protein-encoding gene from the three Cn varieties, which may provide a framework for future studies on the role of Cu,Zn SOD in Cn pathogenesis.
Dynamic properties of bovine Cu,Zn superoxide dismutase from crystallographic data
1998, Inorganica Chimica Acta
The Cu,Zn superoxide dismutase (SOD) enzyme from bovine erythrocytes has been the subject of several crystallographic studies over the last fifteen years. Nine crystal structures in space groups C2, P2₁2₁2₁ and C222₁ have been refined at high resolution and are available from the Protein Data Bank and our laboratory. We have performed a comparative analysis based on the experimentally determined structural variability in order to study the dynamics of the enzyme in terms of molecular flexibility. We have been able to show that information on molecular motions of different types occurring on different timescales may be obtained from the data by comparing thermal motion analysis with that of backbone Cα fluctuations. We have found that the most mobile parts of Cu,Zn SOD are located in turns and loops and that there is a strict correlation between thermal motion and flexibility of the molecule, with the only exception of the two residues Asp11 and Glu38. The analysis of crystal polymorphism established the existence of a quite strict correlation between crystal contacts and mobility in space group C2, while in P2₁2₁2₁ and C222₁ the opposite was observed. Comparison with the results of molecular dynamics simulation revealed a good agreement between the two approaches although some differences emerge when active site residues are considered. The results of this work indicate the feasibility of an ‘experimental’ approach to the study of protein dynamics.
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1998, Advances in Inorganic Chemistry
This chapter discusses structure and properties of copper–zinc superoxide dismutases. The superoxide radical anion is physiologically produced in controlled amounts in animals and plants from the one-electron reduction of dioxygen occurring in several metabolic pathways. Under pathological conditions, large amounts of superoxide and related active oxygen species are released. Thus, a twofold aspect of superoxide effects on living organisms emerges from the available experimental data. The actual role of superoxide dismutase enzymes in living organisms needs to be reconsidered in view of this wider framework. A natural monomeric species has been isolated from the periplasmic space of an Escherichia coli strain. This CuZnSOD displays a catalytic activity comparable to that of bovine SOD, but is highly sensitive to proteases. A comparison of sequences of different Cu₂Zn₂SODs shows that amino acids are invariant, and that most of them are residues close to or part of the active site.
Crystal structure of yeast Cu,Zn superoxide dismutase. Crystallographic refinement at 2.5 Å resolution
1992, Journal of Molecular Biology
The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 Å resolution. The enzyme crystallizes in the P2₁2₁2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 Å resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 Å from the ideal bond lengths, and an average B-factor value of 29.9 Å².
A dimeric molecule of the enzyme is composed of two identical subunits related by a noncrystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded β-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 β-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c β-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 β-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu²⁺ ligands show an uneven distortion from a square plane, while Zn²⁺ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 Å away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme.
The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.
The β-barrel of the bovine enzyme is closed at opposite ends by apolar interactions involving Leu104 and Leu36; the latter is substituted by Ser36 in the yeast enzyme. Three concerted substitutions Ala87 → Thr, His41 → Arg and Glu119 → Ala allow for the formation of a “plug” for the β-barrel by means of a hydrogen bonding scheme.
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1992, Journal of Molecular Biology
Equipotential lines were calculated, using the Poisson-Boltzmann equation, for six Cu,Zn superoxide dismutases with different protein electric charge and various degrees of sequence homology, namely those from ox, pig, sheep, yeast and the isoenzymes A and B from the amphibian Xenopus laevis. The three-dimensional structures of the porcine and ovine superoxide dismutases were obtained by molecular modelling reconstruction using the structure of the highly homologous bovine enzyme as a template. The three-dimensional structure of the evolutionary distant yeast Cu,Zn superoxide dismutase was recently resolved by us, while computer-modelled structures are available for X. laevis isoenzymes. The six proteins display large differences in the net protein charge and distribution of electrically charged surface residues but the trend of the equipotential lines in the proximity of the active sites was found to be constant in all cases. These results are in line with the very similar catlytic rate constants experimentally measured for the corresponding enzyme activities. This analysis shows that electrostatic guidance for the enzyme-substrate interaction in Cu,Zn superoxide dismutases is related to a spatial distribution of charges, arranged so as to maintain, in the area surrounding the active sites, an identical electrostatic potential distribution, which is conserved in the evolution of this protein family.
Progress in the structure and catalysis mechanism of MnSOD and FeSOD
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Crystallographic characterization and three-dimensional model of yeast Cu,Zn superoxide dismutase

Abstract

J. Mol. Biol.

J. Biol. Chem.

Biochem. Biophys. Res. Com.

J. Mol. Biol.

J. Biol. Chem.

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CRC Biochem.

J. Biol. Chem.