Tryptophan Residues Flanking the Second Transmembrane Helix (TM2) Set the Signaling State of the Tar Chemoreceptor

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• 作者：Roger R. Draheim ; Arjan F. Bormans ; Run-zhi Lai ; and Michael D. Manson
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：February 1, 2005
• 年：2005
• 卷：44
• 期：4
• 页码：1268 - 1277
• 全文大小：256K
• 年卷期：v.44,no.4(February 1, 2005)
• ISSN：1520-4995

文摘

The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster inpatches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellarrotation. The functional domains of a receptor are physically separated by the cell membrane.Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediatessignaling and adaptation. These two domains communicate through the second transmembrane helix (TM2)that connects them. In the high-abundance receptors Tar and Tsr, TM2 is flanked by tryptophan residues,which should localize preferentially to the interfacial zone between the polar and hydrophobic layers ofthe phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 ofTar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192Aprotein retains full activity in vivo and in vitro, but it increases the K_i for aspartate in the in vitro assay3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leadsto an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changesseen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrudefarther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similardisplacement. Moving Trp to each position from residue 206 to residue 212 generated a wide variety ofTar signaling states that are generally consistent with the predictions of the piston model of transmembranesignaling. None of these receptors was completely locked in one signaling mode, although most showedpronounced signaling biases. Our findings suggest that the Trp residues flanking TM2, especially Trp-209, are important in setting the baseline activity and ligand sensitivity of the Tar receptor. We alsoconclude that the Tyr-210 residue plays at least an auxiliary role in this control.