| Description |
The chemoreceptor Tsr enables Escherichia coli to track serine gradients in its environment. Binding of serine to the periplasmic domain propagates a signaling conformational change through other domains of the chemoreceptor molecule to modulate the activity of CheA, a kinase associated with the cytoplasmic tip of Tsr. Changes in CheA activity influence the rotational behavior of the cell's flagellar motors. My work focused on two Tsr domains involved in CheA control; the TM2 transmembrane helix connects the periplasmic serine-binding domain to the cytoplasmic kinase control domain, and influences the packing stability of the adjoining HAMP domain via a five- residue control cable. The HAMP domain relays input signals to the kinase control domain; E248, a HAMP domain residue, is critical for the function of the HAMP domain. To analyze the role of E248 in HAMP domain function, I constructed and characterized mutant chemoreceptors with various amino acid replacements at this residue. The E248 alterations abrogated function of the chemoreceptor, either by locking it in the "kinase- off" (E248A, E248G, E248S, E248W) or the "kinase-on" (E248R, E248Q) output state. These results suggest that E248 may be optimal for intermediate stability of the HAMP domain. I also constructed mutant chemoreceptors with one-residue amino acid insertions at various locations within the TM2 domain to assess its structural interactions with the control cable and the HAMP domain. TM2 insertions shifted Tsr output towards the "kinase-on" state, whereas insertions within the control cable generally shifted the chemoreceptor towards the "kinase-off" state. These results suggested a helix-clutch mechanism for transmembrane siglnaling by the Tsr chemoreceptor. |
