Structure of Escherichia coli tyrosine kinase Etk reveals a novel activation mechanism.
While protein tyrosine (Tyr) kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. The inner-membrane Wzc/Etk protein belongs to the bacterial PTK family, which has an important function in regulating the polymerization and transport of virulence-determining capsular polysaccharide (CPS). The kinase uses a unique two-step activation process involving intra-phosphorylation of a Tyr residue, although the molecular mechanism remains unknown. Herein, we report the first crystal structure of a bacterial PTK, the C-terminal kinase domain of Escherichia coli Tyr kinase (Etk) at 2.5-A resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting mass spectrometric evidence of Etk, a unique activation mechanism is proposed that involves the phosphorylated Tyr residue, Y574, at the active site and its specific interaction with a previously unidentified key Arg residue, R614, to unblock the active site. Both in vitro kinase activity and in vivo antibiotics resistance studies using structure-guided mutants further support the novel activation mechanism.
Lee
Daniel C
DC
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Zheng
Jimin
J
She
Yi-Min
YM
Jia
Zongchao
Z
D000595:Amino Acid Sequence; D001692:Biological Transport; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D055672:Static Electricity; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D029968:Escherichia coli Proteins; C120614:Etk protein, E coli; D008565:Membrane Proteins; D050505:Mutant Proteins; D011135:Polysaccharides, Bacterial; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases