Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation 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 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 neurotoxins, and that its covalent modification primarily modulates the protein thermostability.
Blanes-Mira
C
C
Centro de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain.
Ibañez
C
C
Fernández-Ballester
G
G
Planells-Cases
R
R
Pérez-Payá
E
E
Ferrer-Montiel
A
A
D001905:Botulinum Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002942:Circular Dichroism; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D010766:Phosphorylation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D019061:src-Family Kinases; D001905:Botulinum Toxins; C096326:botulinum toxin type E