Structural Organization of the Protein-tyrosine Autokinase Wzc within Escherichia coli Cells

Abstract

Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.

Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

“…However, the data are entirely consistent with the existence of a complex, minimally involving Wza and Wzc. These results differ from those obtained during attempts to cross-link Wzc and Wza in the E. coli K-12 colanic acid (EPS) system using formaldehyde (16). The reason(s) for the difference in results are unknown.…”

“…One attractive explanation for these similar phenotypes is some type of feedback regulatory mechanism, potentially involving an enzyme complex. Wzc provides a good candidate for interaction with the OM in a complex because of its large periplasmic domain (16) and the putative coiled-coil structures in this domain (36). The coiled-coil region is not involved in the known oligomerization of Wzc (14) but could certainly participate in interactions with other proteins.…”

“…One candidate for an IM interaction partner for Wza is Wzc. The Wzc protein is essential for capsule assembly and the close homolog Wzc from E. coli K-12 contains a large periplasmic loop (16); the corresponding region in Wzc from the K30 cps locus comprises 374 amino acids. The periplasmic loop is predicted to be mainly ␣-helical and contains a predicted coiledcoil motif (36), a feature that could be involved in proteinprotein interactions.…”

“…In previous cross-linking experiments, Wzc was shown to oligomerize independent of Wza (13,16). To determine whether the interaction between Wza and Wzc is essential for the formation of stable Wza multimers, Wza was expressed in the wzc-null strain, E. coli CWG285.…”

PhpA, a tyrosine phosphatase of Myxococcus xanthus, is involved in the production of exopolysaccharide

“…These BY kinases share no sequence homology with protein tyrosine kinases in eukaryotes. BY kinases from Gramnegative bacteria are usually large membrane-spanning multi-domain proteins composed of an N-terminal periplasmic region and a C-terminal cytosolic tyrosine kinase domain (Doublet et al, 2002). In Gram-positive bacteria, these BY kinases are often split into two distinct proteins, a periplasmic domain and a cytoplasmic protein kinase.…”