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

Abstract

The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediates signaling 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 of the phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 of Tar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192A protein retains full activity in vivo and in vitro, but it increases the K(i) for aspartate in the in vitro assay 3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leads to an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changes seen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrude farther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similar displacement. Moving Trp to each position from residue 206 to residue 212 generated a wide variety of Tar signaling states that are generally consistent with the predictions of the piston model of transmembrane signaling. None of these receptors was completely locked in one signaling mode, although most showed pronounced 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 also conclude that the Tyr-210 residue plays at least an auxiliary role in this control.

Mutational Analysis of the Control Cable That Mediates Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

“…2B). The sharp-edge phenotype has been seen previously in Tar mutants (22,68) and is most likely a symptom of the elevated methylation status of ATT-mimic receptors. Extensively methylated Tsr molecules should have a reduced dynamic range of serine sensing (38,39), so the sharp cell band probably reflects congregation of the migrating cells in a narrow concentration range at the high end of the serine gradient.…”

“…1) (9,22,34). A five-residue segment joins F212 to L218, the first critical residue of the HAMP AS1 helices ( Fig.…”

Topology and Boundaries of the Aerotaxis Receptor Aer in the Membrane ofEscherichia coli

“…Notably, residues 163 and 206 are tryptophans in native Aer. Tryptophans are well known for stabilizing membraneaqueous boundaries (13,67). Given their hydrophobic nature, it is likely that these native tryptophan residues reside at the membrane boundary interface rather than protrude into the cytosol, as occurred for their cysteine replacements.…”

“…The loop itself does not appear to be involved in signal transduction. Whether the function of the membrane anchor is to localize Aer to the membrane, maintain registry between N-terminal PAS and C-terminal signaling regions, or be actively involved in signaling like the MCPs (13,42) remains to be determined.…”

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…Such experiments have been done by introducing or moving charged or aromatic residues near the periplasmic and cytoplasmic membrane-water interfaces of the signaling helix with the aim of creating electrostatic or hydrophobic forces that would slide the helix along its long axis perpendicular to the membrane [51][52][53]. In support of the piston mechanism, substitutions expected to drive the signaling helix towards the cytoplasm shifted the receptor to the off-state of lower kinase activation and higher methylation rate, whereas substitutions driving movement towards the periplasm shifted the receptor to the onstate, displaying higher kinase activation and lower methylation rate [51][52][53]. These results indicate that native residues near the membrane-water interface in the signaling helix have an important role in modulating receptor on-off bias by stabilizing that helix in a proper transmembrane register.…”