Subunit interaction in extracellular superoxide dismutase: Effects of mutations in the N‐terminal domain

• Published in: Protein science Vol. 6; no. 11; pp. 2350 - 2358
• Main Authors: Stenlund, Peter, Tibell, Lena A. E., Andersson, Dick
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.11.1997
• Subjects: Amino Acid Sequence; carbonic anhydrase II; Circular Dichroism; extracellular superoxide dismutase; heterologous expression; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Binding - genetics; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; site‐directed mutagenesis; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; subunit interaction; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; tetramer contact area
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560061108

Human extracellular superoxide dismutase (hEC‐SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC‐SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X‐ray structural determination have failed, the three‐dimensional structure of hEC‐SOD is unknown. This means that alternative strategies for structural studies are needed. The N‐terminal domain of EC‐SOD has already been studied using the fusion protein FusNN, comprised of the 49 N‐terminal amino acids from hEC‐SOD fused to human carbonic anhydrase (HCAII). The N‐terminal domain in this fusion protein forms a well‐defined three‐dimensional structure, which probably contains α‐helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site‐directed mutagenesis in combination with size‐exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic a‐helix (formed by residues 14‐32) in the N‐terminal domain is essential for the subunit interaction.