Structure of Escherichia coli tyrosine kinase Etk reveals a novel activation mechanism

Abstract

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-Å 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.

Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways

“…Single-particle EM studies of detergent-solubilized Wzc showed that it is a tetramer (14,16). However, members of this subfamily carry an extended cytoplasmic kinase domain (not found in other PCP proteins) that, in isolated form, is an octamer in crystal structures (41,42). It is unclear whether the difference in oligomeric states reflects the absence of constraining transmembrane and periplasmic domains in the crystal structure.…”

Bacterial tyrosine kinases: evolution, biological function and structural insights

“…More recently, the crystal structures of the cytoplasmic domains of Etk, a paralogue of Wzc in E. coli [59], and of the orthologue CapB from the Firmicute Staphylococcus aureus [53] have been solved. Both structures displayed a Rossman fold typical of P-loop proteins [51].…”

“…Etk, like its paralogue Wzc, possesses outside its C-terminal Y-cluster an additional tyrosine that had been suggested to be involved in an intramolecular phosphorylation step, preceding transautophosphorylation of the Y-cluster [52]. The analysis of the Etk structure suggested that that this tyrosine points into the active site, and once phosphorylated it could flip out of the active site to allow ATP binding and subsequent phosphorylation of the Y-cluster [59,62]. However, its orientation seems quite incompatible with phosphate transfer, and this residue is not conserved among all BY-kinases from Proteobacteria.…”

Comparative Analysis of the Tyr-Kinases CapB1 and CapB2 Fused to Their Cognate Modulators CapA1 and CapA2 from Staphylococcus aureus

“…In this organization, the Y-cluster of each subunit is bound to the active site of the neighboring subunit for trans-autophosphorylation. On the other hand, the crystal structure of the cytoplasmic kinase domain of the phosphorylated form of the Wzc orthologue Etk from E. coli [16] (PDB ID 3CIO) showed that it forms a monomer with a flexible and unstructured Y-cluster. Altogether, it is proposed that cyclic autophosphorylation and dephosphorylation of BY-kinases regulates association/dissociation of the octamer and thus the functioning of the capsule assembly machinery [4].…”