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AbstractSuperoxide and its reactive byproducts are generated in large amounts as defense mechanism by neutrophils against pathogenic microbes. Superoxide is produced by a membrane-bound multi-subunit NADPH oxidase that transfers electrons from NADPH to molecular oxygen. In this study, we are investigating the p67 phox C-terminal SH3B domain which binds to p47phox proline-rich region using the yeast two-hybrid system combine with random mutagenesis and site-directed mutagenesis to identify mutations of boundary sequence in p67phox C-terminal SH3B domain. The amino acid P55 has been changed to Ile, Gly, Val, Trp, Asn, Glu, His and Phe or produce stop codon, resulting in the binding to p47phox proline-rich region activity was completely abolished. Changing F58 to Tyr has no effect of this binding, while change to stop codon completely abolished the activity. Furthermore, the amino acid V59 is replaced by stop codon, the activity was also completely abolished. The amino acid E60 is replaced by stop codon shown no effect of binding. Taken together, the resultsOverexpressing of the V59stop and E60stop two mutant of p67phox C-terminal SH3B domain in the bacterial pET expressing system. The product of the E60stop fusion protein is showing unstable compare to the product of V59stop fusion protein. This suggests that this two fusion protein may exist structure difference in their conformation. In vitro binding assays show that both two forms have binding activity of target GST-p47 differ in the yeast two-hybrid system. This suggests that in the bacterial expression sThe mutation of V59 to Phe﹑Ser﹑Gly or Ile did not effect the ability of the p67phox C-terminal SH3B domain to bind p47phox , which shown this residue may not a conserved residue . However, replaced by Leu or Glu result in loss of in interaction ability. We speculate that this residue may play its specificity for protein-protein interactions in phagocyte NADPH oxidase (since four SH3 domains existing in p67 phox and p47phox of NADPH oxidase, only p67phox C-terminal SH3 domain containing V59, the other three
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