Octameric Kinase Wzc in E. coli
Created by: Sarah Davidson
According to PDB, the protein Wzc (PDB-ID: 3LA6) is an octameric kinase consisting of sixteen subunits of 286 amino acid residues each, for a total of 4576 total residues. According the PDB, each subunit has an average of 11 alpha-helices and 10 beta-sheets.Together, the subunits form two octameric rings. Wzc is a member of the capsular exopolysaccharide family, which includes proteins involved in exopolysaccharide biosynthesis. Specifically, the protein Wzc is involved in the synthesis of the exopolysaccharide colanic acid.
Wzc is a BY-kinase, which refers to a group of bacterial tyrosine kinases. These kinases have no eukaryotic homologues (Jadeau, Bechet, et al). Wzc belongs to the superfamily of P-loop NTP-ases. P-loop NTP-ases contain a conserved Walker A and Walker B motifs (Aravind, Iyer, et al). The Walker A motif is involved with the binding of a triphosphate group of an NTP, while the Walker B motif is involved with NTP hydrolysis (Arayind, Iyer, et al). The Walker A and B motifs make up the active site of the Wzc kinase (Lacour, Bechet, et al).
The protein Wzc can associate with the protein Wza, another octameric protein, to form a structure that can span both the inner and outer membranes of the gram-negative bacteria (Bechet, Gruszczyk, et al). The role of Wzc in transport across the membrane is uncertain, and the Wzc ring might be filled by the lipid bilayer (Bechet, Gruszczyk, et al). The protein Wzc has two octameric rings that interact (Bechet, Gruszczyk, et al). In addition, the subunits within each octameric ring interact through the binding of the Tyr-715 residue to the active site of the neighboring subunit (Bechet, Gruszczyk, et al). It is thought that phosphorylation of this residue in each subunit causes the complex to dissociate into monomers, as once the residue has been phosphorylated, it can no longer interact as well with the active site of the neighboring subunit (Bechet, Gruszczyk, et al). The octameric structure is also stabilized by a EX2RX2R motif (Bechet, Gruszczyk, et al). The protein kinase Wzc can also be phosphorylated on resides Tyr-708, Tyr-710, Tyr-711 and Tyr-713 (Bechet, Gruszczyk, et al). The ADP molecule fits in the active site through several interactions. A calcium ion interacts with the hydroxyl group of Thr-541 and the beta phosphate of the ADP molecule (Bechet, Gruszczyk, et al). The beta phosphate also interacts with nitrogen atoms (blue is the nitrogen) in residues 537, 538, and 539 (Bechet, Gruszczyk, et al). The alpha phosphate interacts with the hydroxyl group of Tyr-569, and the hydroxyl groups of the ribose group interact with Asp-480 (Bechet, Gruszczyk, et al). The adenine group can hydrogen bond with Asn-696, Pro-471, and Ser-473 as well (Bechet, Gruszczyk, et al). An RK-cluster (identified as residues 473-496) is also thought to be essential for the kinase activity of tyrosine kinase Wzc (Bechet, Gruszczyk, et al).
The protein showed similarities to the chimerical mutant capabk55m protein in the bacteria Staphylococcus aureus. Both the chimerical mutant capabl55m protein and the octameric kinase domain of the Wzc protein are BY-kinases, which means both shared the conserved Walker A and Walker B motifs, which further means that both proteins are involved with NTP binding and hydrolysis (Bechet, Gruszczyk, et al).
In the gram-negative bacteria E. coli, the tyrosine kinase Wzc phosphorylates UDP-glucose dehydrogenase, along with another BY-kinase Etk. The phosphorylation of the UDP-glucose dehydrogenase allows it to produce UDP-glucuronic acid, which is a precursor for many bacterial polysaccharides, including colanic acid (Lacour, Bechet, et al). According to Chris Whitfield, the main purpose of colanic acid is when the bacterium is outside a host. Colanic acid is thought to be involved with biofilm formation on abiotic surfaces (Whitfield). A point mutation of D480A resulted in a 90% decrease in autokinase activity, and point mutations of K489A, R490A, K492A, and Q493A resulted in a 50% decrease in autokinase activity. The mutations in Asp-480, Lys-489, Arg-490, and Lys-492 all demonstrated a sharp decrease in the production of colanic acid, indicating that these mutations disrupted Wzc's role in that process (Bechet, Gruszczyk, et al).
The polysaccharide capsule found in bacteria is known to be a virulence factor, since the capsular polysaccharides can protect the bacteria against components of the human immune system (Bechet, Gruszczyk, etc). Colanic acid itself is not involved in the virulence of bacteria (Whitfield), but Wzc is a BY-kinase, and BY-kinases do not resemble eukaryotic tyrosine kinases (Bechet, Gruszczyk, et al), which means that BY-kinases could be a possible target for drugs to treat bacterial infections with minimal harm to the infected person.