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


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 neurotoxins, and that its covalent modification primarily modulates the protein thermostability. Study holds ProTherm entries: 10878, 10879 Extra Details: clostridial neurotoxins; tyrosine-specific protein kinases;,recombinant catalytic domain; alpha-helical content; tyrosine

Submission Details

ID: RhGTrvrx

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:41 p.m.

Version: 1

Publication Details
Blanes-Mira C;Ibañez C;Fernández-Ballester G;Planells-Cases R;Pérez-Payá E;Ferrer-Montiel A,Biochemistry (2001) Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue. PMID:11329292
Additional Information

Structure view and single mutant data analysis

Study data

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Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1T3C 2004-06-29 1.9 Clostridium botulinum type E catalytic domain E212Q mutant
1T3A 2004-06-29 2.16 Crystal structure of Clostridium botulinum neurotoxin type E catalytic domain
1ZKW 2005-06-28 2.17 Crystal structure of Arg347Ala mutant of botulinum neurotoxin E catalytic domain
1ZL6 2005-06-28 2.4 Crystal structure of Tyr350Ala mutant of Clostridium botulinum neurotoxin E catalytic domain
1ZKX 2005-07-05 2.52 Crystal structure of Glu158Ala/Thr159Ala/Asn160Ala- a triple mutant of Clostridium botulinum neurotoxin E catalytic domain
1ZL5 2005-07-05 2.6 Crystal structure of Glu335Gln mutant of Clostridium botulinum neurotoxin E catalytic domain
1ZN3 2005-07-05 2.6 Crystal structure of Glu335Ala mutant of Clostridium botulinum neurotoxin type E
3FFZ 2008-12-16 2.65 Domain organization in Clostridium butulinum neurotoxin type E is unique: Its implication in faster translocation

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.9 Botulinum neurotoxin type E P30995 BXE_CLOBU
100.0 Botulinum neurotoxin type E Q00496 BXE_CLOBO