Direct evidence that the carboxyl‐terminal sequence of a bacterial chemoreceptor is an unstructured linker and enzyme tether

Abstract

Sensory adaptation in bacterial chemotaxis involves reversible methylation of specific glutamyl residues on chemoreceptors. The reactions are catalyzed by a dedicated methyltransferase and dedicated methylesterase. In Escherichia coli and related organisms, control of these enzymes includes an evolutionarily recent addition of interaction with a pentapeptide activator located at the carboxyl terminus of the receptor polypeptide chain. Effective enzyme activation requires not only the pentapeptide but also a segment of the receptor polypeptide chain between that sequence and the coiled-coil body of the chemoreceptor. This segment has features consistent with a role as a flexible and presumably unstructured linker and enzyme tether, but there has been no direct information about its structure. We used site-directed spin labeling and electron paramagnetic resonance spectroscopy to characterize structural features of the carboxyl-terminal 40 residues of E. coli chemoreceptor Tar. Beginning~35 residues from the carboxyl terminus and continuing to the end of the protein, spectra of spin-labeled Tar embedded in native membranes or in reconstituted proteoliposomes, exhibited mobilities characteristic of unstructured, disordered segments. Binding of methyltransferase substantially reduced mobility for positions in or near the pentapeptide but mobility for the linker sequence remained high, being only modestly reduced in a gradient of decreasing effects for 10-15 residues, a pattern consistent with the linker providing a flexible arm that would allow enzyme diffusion within defined limits. Thus, our data identify that the carboxyl-terminal linker between the receptor body and the pentapeptide is an unstructured, disordered segment that can serve as a flexible arm and enzyme tether.

Bacterial Chemoreceptor Dynamics: Helical Stability in the Cytoplasmic Domain Varies with Functional Segment and Adaptational Modification

“…Two of these correlation times were for spin labels on the cytoplasmic domain of Tar denatured in 4 M urea. The third was for a spin label near the end of the natively disordered, 35-residue flexible arm at the extreme carboxyl terminus of Tar [42]. Fitting these spectra computationally identified two components with different mobilities, a predominant (63 to 93%), highly mobile state and a minority (7 to 37%) less mobile state.…”

“…As spectra from disordered backbones, we used those described in the previous paragraph, two for spin labels on urea-denatured Tar and one for a spin label on the disordered, Tar carboxyl-terminal flexible arm [42] (Fig. 6B, inset).…”

“…These spectra were for Tar carrying a spin-labeled cysteine at position 478 or 483 denatured in 4M urea, and Tar spin labeled at position 543 in the 35-residue disordered flexible arm at the carboxyl terminus of Tar [42]. …”

Bacterial chemoreceptor dynamics correlate with activity state and are coupled over long distances

“…The inverse of the central line width (ΔH −1 ) and overall spectral breadth (31) were used to assess the mobility of TM1 in the ligandfree chemoreceptor Trg (43) and the C-terminal CheR-targeting peptide of Tar (44). Our CW-ESR data on the HAMP KCM module indicate an equilibrium of two conformational states.…”

Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array

“…The appended N terminal residues were modeled as an alpha helix. The appended C-terminal residues are known to be unstructured, 31 forming a flexible tail that binds the CheR methyltransferase for methylation of sites in the same and nearby CF subunits. 32 Database values for random coil chemical shifts 33 were used for these C terminal residues in the final predicted spectra.…”