Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function [Protein Structure and Folding]

July 7th, 2014 by Roach, E. J., Kimber, M. S., Khursigara, C. M.

FtsZ is an essential cell division protein in Escherichia coli and its localization, filamentation and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. FtsZ- associated proteins (Zaps) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The X-ray crystallographic structure of E. coli ZapA (EcZapA) was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in EcZapA were modified using site- directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared to wild-type (WT) ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ∆zapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling, and which ultimately affect Z-ring formation in dividing cells.
  • Posted in Journal of Biological Chemistry, Publications
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