The Structure of Human Extracellular Copper–Zinc Superoxide Dismutase at 1.7 Å Resolution: Insights into Heparin and Collagen Binding

https://doi.org/10.1016/j.jmb.2009.03.026Get rights and content

Summary

Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 Å resolution. The overall subunit fold and the subunit–subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu–Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.

Introduction

The superoxide radical is the oxygen radical formed in the greatest amount in the metabolism of molecular oxygen. It is degraded by superoxide dismutases (SODs),1 of which there are three isoenzymes in mammals. Copper–zinc SOD (SOD1) occurs in the cytosol,1 the intermembrane space of mitochondria,2 and the nucleus,3 while manganese SOD (SOD2) is localized to the mitochondrial matrix.2 Extracellular superoxide dismutase (SOD3) is secreted to the extracellular space where it performs the same enzymatic reaction as that of SOD1, namely, the dismutation of superoxide to hydrogen peroxide and oxygen,4, 5 and at a similar rate (∼ 109 M 1 s 1 per Cu atom). Because the superoxide radical penetrates membranes poorly, the three SOD isoenzymes are generally assumed to have distinct roles in the body.6

SOD3 is a homotetrameric Cu- and Zn-containing glycoprotein, which shows affinity for heparin and other sulfated glycosaminoglycans.4, 7 The major proportion in the body exists anchored to heparan sulfate proteoglycans† in the tissue interstitium and on cell surfaces.8 SOD3 also shows affinity for collagen type 19 and for fibulin-5.10 SOD3 occurs in extracellular fluids such as plasma, lymph, cerebrospinal fluid, and seminal plasma.4, 11 While SOD1 and SOD2 are ubiquitously expressed in the body, SOD3 is synthesized by a more limited number of cell types.12 The levels of SOD3 are particularly high in blood vessel walls and in the lungs. Studies in mice lacking SOD3 suggest that the enzyme plays multiple roles, including protection of the lung against hyperoxia,13 preservation of nitric oxide by limiting the superoxide available for formation of peroxynitrite,14 reduction of angiotensin-2-induced hypertension15, promotion of neovascularization,16 and retention of memory.17 The importance of the tissue anchoring is demonstrated by the increased risk of myocardial infarctions and stroke in individuals that carry a common polymorphism, R213G, which reduces the heparin affinity of SOD3.18 Unlike wild-type human SOD3, this mutant also fails to reduce the blood pressure when expressed in spontaneously hypertensive rats.19 SOD3 is synthesized with an 18-amino-acid-long signal peptide, which after cleavage leaves a 222-amino-acid-long mature subunit.20 The central part of the sequence (residues 91–194) was predicted to have relatively strong similarities with human SOD1. The N-terminal end appears to harbour parts important for the tetramer interface21 and contains the glycosylation site, Asn89.22, 23 SOD3 is the only glycosylated SOD isoenzyme. While a specific biological function for the carbohydrate has not been identified, the nonglycosylated protein is significantly less soluble.22 A cluster of positively charged residues in the C-terminal end confers the heparin affinity.24 Despite extensive efforts, the structure determination of SOD3 has remained a challenge. We report here the first structure of human SOD3 at 1.7 Å resolution. The shape of the homotetramer allows us to propose the likely heparin- and collagen-binding sites, located between the two ‘SOD1-like’ dimers.

Section snippets

Quality of the model

The structure of recombinant human SOD3 has been determined to a resolution of 1.7 Å. The final R-factor and Rfree are 15.1% and 18.5%, respectively (Table 1). The crystal is in the P21 space group with one tetramer in the asymmetric unit. The average B-factor for the model was 29 Å2. The N-terminal (residues 1–37) and C-terminal (residues 206–222) portions of the protein were not modelled due to lack of clear density within the 2Fo  Fc and Fo  Fc maps. The final model contains 5108 protein

Protein production and crystallisation

Recombinant SOD3 was expressed in Chinese hamster ovary cells and isolated as previously described,51 with a heparin–Sepharose chromatography included, which ensures that the C-terminus is present. Crystals were obtained using the hanging-drop vapour-diffusion technique. Droplets containing protein (20 mg/ml) with 0.1 M potassium thiocyanate, 0.05 M Bis–Tris propane (pH 7.5), and 10% (w/v) PEG (polyethylene glycol) 3350 were equilibrated over wells containing 0.2 M potassium thiocyanate, 0.1 M

Acknowledgements

We acknowledge support from the Biotechnology and Biological Sciences Research Council UK and the Swedish Science Council, and access to facilities at the Science and Technology Facilities Council Synchrotron Radiation Source at Daresbury Laboratory UK.

References (60)

  • HoughM.A. et al.

    Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119–Leu142 loop region for catalytic function

    J. Mol. Biol.

    (2000)
  • StrangeR.W. et al.

    The structure of holo and metal-deficient wild-type human Cu, Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis

    J. Mol. Biol.

    (2003)
  • RakhitR. et al.

    Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis

    J. Biol. Chem.

    (2002)
  • BorgstahlG.E.O. et al.

    The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4 helix bundles

    Cell

    (1992)
  • HallewellR.A. et al.

    Genetically engineered polymers of human CuZn superoxide dismutase

    J. Biol. Chem.

    (1989)
  • AdachiT. et al.

    Interactions between human extracellular superoxide dismutase C and sulphated polysaccharides

    J. Biol. Chem.

    (1989)
  • BoissinotM. et al.

    Rational design and expression of a heparin-targeted human superoxide dismutase

    Biochem. Biophys. Res. Commun.

    (1993)
  • OtwinowskiZ. et al.

    Processing of X-ray diffraction data collected in oscillation mode

  • LamzinV.S. et al.

    Automated refinement for protein crystallography

  • ChangL.Y. et al.

    Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes

    J. Cell Biol.

    (1988)
  • MarklundS.L.

    Human copper-containing superoxide dismutase of high molecular weight

    Proc. Natl. Acad. Sci. USA

    (1982)
  • WinterbournC.C. et al.

    Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical

    J. Clin. Invest.

    (1987)
  • KarlssonK. et al.

    Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans

    Biochem. J.

    (1988)
  • MarklundS.L.

    Extracellular superoxide dismutase in human tissues and human cell lines

    J. Clin. Invest.

    (1984)
  • NguyenA.D. et al.

    Fibulin-5 is a novel binding protein for extracellular superoxide dismutase

    Circ. Res.

    (2004)
  • PeekerR. et al.

    Superoxide dismutase isoenzymes in human seminal plasma and spermatozoa

    Mol. Hum. Reprod.

    (1997)
  • MarklundS.L.

    Expression of extracellular superoxide dismutase by human cell lines

    Biochem. J.

    (1990)
  • CarlssonL.M. et al.

    Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia

    Proc. Natl Acad. Sci. USA

    (1995)
  • BeckmanJ.S. et al.

    Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide

    Proc. Natl Acad. Sci., USA

    (1990)
  • JungO. et al.

    Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice

    Circ. Res.

    (2003)
  • Cited by (99)

    • Superoxide dismutases inhibitors

      2023, Metalloenzymes: From Bench to Bedside
    • SOD1, more than just an antioxidant

      2021, Archives of Biochemistry and Biophysics
      Citation Excerpt :

      SOD activity relies on a specific catalytic metal ion, which could be either manganese (MnSOD), iron (FeSOD), nickel (NiSOD), or copper (Cu/ZnSOD) [2]. These classes of the SOD molecule have evolved in various organisms; in eukaryotes, MnSOD and Cu/ZnSOD are the major isoforms [3–6]. All mammals (including humans) possess three isoforms of superoxide dismutase: Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3).

    View all citing articles on Scopus
    View full text