Linked Life Data

19634880

Source:http://linkedlifedata.com/resource/pubmed/id/19634880

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
33
pubmed:dateCreated
2009-8-18
pubmed:abstractText
Escherichia coli tyrosine kinase (Etk) regulates the export of pathogenic capsular polysaccharide (CPS) by intermolecularly autophosphorylating its C-terminal tyrosine cluster. The kinase Etk, however, needs to be first activated by the intramolecular phosphorylation of a tyrosine residue, Y574, next to the active site. The recently determined structure of Etk shows that dephosphorylated Y574 blocks the active site and prevents substrate access. After phosphorylation, the negatively charged P-Y574 side chain was previously postulated to flip out to associate with a positively charged R614, unblocking the active site. This proposed activation is unique among protein kinases; however, there is no direct structural evidence in support of this hypothesis. In this paper, we carried out molecular dynamics simulation, mutagenesis, and biochemical analysis to study the activation mechanism of Etk. Our simulation results are in excellent agreement with the proposed molecular switch involving P-Y574 and R614 in the activation of Etk. Further, we show that a previously unidentified residue, R572, modulates the rotation of the P-Y574 side chain through electrostatic interaction, slowing down the opening of the active site. Our enzymatic assays demonstrate that the R572A mutant of Etk possesses significantly increased kinase activity, providing direct experimental support for the unique activation mechanism of Etk. In addition, the simulation of the Etk Y574F mutant predicted short periods of unblocked active site by Y574F, in good agreement with the low kinase activity of this mutant. The C-terminal substrate peptide and the nucleotide cofactor were also docked into the active site, and their implications are discussed.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
25
pubmed:volume
48
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7986-95
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2009
pubmed:articleTitle
New insights into the activation of Escherichia coli tyrosine kinase revealed by molecular dynamics simulation and biochemical analysis.
pubmed:affiliation
Department of Chemistry, Beijing Normal University, Beijing 100875, People's Republic of China.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't