Structural requirements for high-affinity heparin binding: alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase.
An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.
Stenlund
Peter
P
Department of Biochemistry, Umeå University, S-901 87 Umeå, Sweden.
Lindberg
Mikael J
MJ
Tibell
Lena A E
LA
D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D019281:Dimerization; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry*; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
C037405:heparin-sepharose; D006493:Heparin; D012685:Sepharose; D013482:Superoxide Dismutase; D000409:Alanine