Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue

Biochemistry
Clara Blanes-MiraAntonio Ferrer-Montiel

Abstract

The catalytic domain of clostridial neurotoxins is a substrate of tyrosine-specific protein kinases. The functional role of tyrosine phosphorylation and also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neurotoxin E (BoNT E) to examine these issues. Bacterially expressed and purified BoNT E catalytic domain was fully active, and was phosphorylated in vitro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the catalytic domain increased the protein thermal stability without affecting its proteolytic activity. Covalent modification of the endopeptidase promoted a disorder-to-order transition, as evidenced by the 35% increment of the alpha-helical content, which resulted in a 4 degrees C increase of its denaturation temperature. Site-directed replacement of tyrosine at position 67 completely abolished phosphate incorporation by Src. Constitutively unphosphorylated endopeptidase mutants exhibited functional properties virtually identical to those displayed by the nonphosphorylated wild-type catalytic domain. These findings indicate the presence of a single phosphorylation site in the catalytic domain of clostridial neurotoxi...Continue Reading

References

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Citations

Sep 22, 2006·Infection and Immunity·Fang CaiJames E Keller
Dec 20, 2008·PLoS Pathogens·Audrey FischerMauricio Montal
Jun 15, 2007·Proceedings of the National Academy of Sciences of the United States of America·Audrey Fischer, Mauricio Montal
May 24, 2008·International Journal of Cosmetic Science·C Blanes-MiraA Ferrer-Montiel
Dec 8, 2004·FEBS Letters·Cristina IbañezAntonio Ferrer-Montiel
Dec 17, 2003·Journal of Neurochemistry·Clara Blanes-MiraAntonio Ferrer-Montiel
Apr 21, 2004·Protein Science : a Publication of the Protein Society·Frederico Faria MirandaAurora Martínez
Aug 6, 2004·Protein Expression and Purification·Michael R BaldwinJoseph T Barbieri
Nov 3, 2007·Journal of Immunological Methods·R G A JonesD Sesardic
Apr 7, 2004·The Journal of Biological Chemistry·Cruz Morenilla-PalaoAntonio Ferrer-Montiel

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