Mutationally Altered Signal Output in the Nart (NarX-Tar) Hybrid Chemoreceptor

Abstract

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate and maltose (39), the latter via maltose-binding protein (18), and repellent responses to Ni 2ϩ and Co 2ϩ (45). The other high-abundance chemoreceptor, Tsr, mediates attractant and repellent responses to serine (39) and leucine (45), respectively. Tar and Tsr form homodimers in the presence or absence of ligands (5, 31), and these dimers associate into both homogeneous (19,21,43,46) and mixed (3,25,36,43,46) trimers of dimers. A number of sensor kinases, including NarX and EnvZ, share predicted membrane topology with these chemoreceptors (10, 13).The Tar and Tsr proteins contain two membrane-spanning regions that connect an N-terminal periplasmic ligand recognition domain to a C-terminal cytoplasmic signaling and adaptation domain (23,24). Tar binds aspartate at the dimer interface near the apexes of the periplasmic domains (8,27,28,31), whereas ligand-bound maltose-binding protein binds asymmetrically at the apex of the Tar homodimer (6,16,53). Serine and leucine bind directly to the periplasmic domain of Tsr (23). The cytoplasmic regions of the high-abundance transducers are responsible for transmitting the signal received from the periplasmic region to the CheA kinase (17,30,44). When the chemical environment is homogeneous, CheA activity is at its baseline level, the flagellar motors alternate between counterclockwise (CCW) and clockwise (CW) rotation, and the cell exhibits normal run-tumble motility (37). The addition of attractants, or the removal of repellents, inhibits the activation of CheA by the cognate receptor, thereby decreasing the rate of phosphoryl group transfer from receptor-associated CheA to the response regulator CheY. Lowering the cytoplasmic level of phospho...

A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

“…However, two lines of evidence indicate that the resultant structural asymmetry is not essential to the transmembrane signaling mechanism. First, the nitrate-sensing domain of NarX, which undergoes quasisymmetric conformational changes upon ligand binding (37), mediates chemotactic responses to nitrate gradients in a chimeric chemoreceptor (38,39). Second, the control cable alterations in the present study were necessarily present in both subunits of the receptor dimer, presumably creating symmetric conformational changes in the mutant Tsr molecules.…”

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

“…However, symmetric structural changes (amino acid replacements, disulfide bonds) in the periplasmic sensing domain or TM bundle can also mimic chemoeffector signaling effects (shifted outputs, altered adaptation responses) (6,7,22,23,25,71). Moreover, the hybrid transducer Nart, which carries the periplasmic sensing through the HAMP domains of NarX joined to the methylation and kinase control domains of Tar (64,65), mediates chemotactic responses to nitrate, whose binding causes quasisymmetric piston displacements (19). We conclude that symmetric conformational inputs most likely modulate HAMP signaling by the same mechanism as that used for asymmetric TM2 piston displacements.…”

The S-Helix Determines the Signal in a Tsr Receptor/Adenylyl Cyclase Reporter

“…In several instances it has been demonstrated that between one-and two-component signaling systems individual domains are interchangeable without loss of function (11)(12)(13)(14)(15)(16). In many signal transduction systems, HAMP 2 domains (histidine kinase, adenylyl cyclase, methyl-accepting proteins of chemotaxis, protein-phosphatases (17)), which are four-bundle parallel coiled coils of about 55 residues, are interspaced between sensory and output domains (18 -19).…”