PMID:12672798

Citation	Zhu, Y and Inouye, M (2003) Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein, Tez1. J. Biol. Chem. 278:22812-9
Abstract	Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker. Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype. Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction. Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype. Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response. None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function. In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation. Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input. In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.
Links	PubMed Online version:10.1074/jbc.M300916200
Keywords	Alanine; Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/metabolism; Base Sequence; Binding Sites; Chemoreceptor Cells; DNA Primers; Dimerization; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Genetic Variation; Molecular Sequence Data; Multienzyme Complexes/chemistry; Multienzyme Complexes/genetics; Multienzyme Complexes/metabolism; Mutagenesis; Mutagenesis, Insertional; Polymerase Chain Reaction; Protein Structure, Secondary; Receptors, Cell Surface/chemistry; Receptors, Cell Surface/genetics; Receptors, Cell Surface/metabolism; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/metabolism; Restriction Mapping; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction/physiology
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