Functional analysis of predicted coiled coil regions in the Escherichia coli K-12 O antigen polysaccharide chain length determinant Wzz

Infectious Diseases Research Group, Siebens-Drake Research Building, Department of Microbiology and Immunology, and Department of Medicine, University of Western Ontario, London, Ontario, N6A 5C1, Canada

^* To whom correspondence should be addressed. Email: mvalvano{at}uwo.ca.

	Abstract

Wzz is a membrane protein determining the chain length distribution of the O-antigen lipopolysaccharide (LPS) by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of the Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils we have constructed amino acid replacement mutants in the E. coli K-12 Wzz protein, which were designed in a manner allowing the coiled coils to separate without compromising the helicity of the individual molecules. Mutations in one of these regions, spanning amino acids 108-130 (Region I), were associated with a partial defect in O antigen chain length distribution, while mutations in the region spanning amino acids 209-223 (Region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153-173 (Region II) abolished Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of Region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted as coiled coils are important for Wzz function by maintaining the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.