Arjan F. Bormans

Maltose‐binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L‐aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose‐binding protein (MBP), which in its ligand‐stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP‐binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr‐143, Asn‐145, Gly‐147, Tyr‐149, and Phe‐150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg‐73, Met‐76, Asp‐77, and Ser‐83. These two sets of mutations defined two complementation groups when the defective proteins were co‐expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met‐75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane‐distal apex of the domain and also extended onto one shoulder of the apical region.

Mutationally altered signal output in the Nart (NarX-Tar) hybrid chemoreceptor.

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.

A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli.

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate‐loaded maltose‐binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand‐binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid‐borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (Δtar ). However, when Tar proteins with complementing sets of mutations were co‐expressed from compatible plasmids, the resulting heterodimeric receptors enabled Δtar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the ‘same’ orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the ‘opposite’ orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the ‘opposite’ binding orientation. When aspartate and MBP bind in the ‘same’ orientation, they compete for signalling through one subunit. In the wild‐type Tar dimer, aspartate and MBP can bind in either the ‘same’ or the ‘opposite’ orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar+ cells.