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

The catalytic domain of clostridial neurotoxins is a substrate of tyrosine- specific protein kinases. The functional role of tyrosine phosphorylation a nd also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neu rotoxin E (BoNT E) to examine these issues. Bacterially expressed and purif ied BoNT E catalytic domain was fully active, and was phosphorylated in vit ro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the cat alytic 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 al pha -helical content, which resulted in a 4 degreesC increase of its denatu ration temperature. Site-directed replacement of tyrosine at position 67 co mpletely abolished phosphate incorporation by Src. Constitutively unphospho rylated endopeptidase mutants exhibited functional properties virtually ide ntical to those displayed by the nonphosphorylated wild-type catalytic doma in. These findings indicate the presence of a single phosphorylation site i n the catalytic domain of clostridial neurotoxins, and that its covalent mo dification primarily modulates the protein thermostability.