Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues

Abstract

Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to be an efficient way to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was shown to be the major determinant governing signal output. However, these previous studies failed to determine whether moving the aromatic residues within TM2 of Tar or EnvZ was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of TM2. Here, we have employed a glycosylation-mapping technique to demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 is sufficient to displace the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. In summary, our results support recent CG-MD simulations with aromatically tuned Tar segments that demonstrated the Trp-Tyr tandem is sufficient to displace TM2 within a membrane. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. We suggest that differences observed between the behavior of helices is due to the inherently different properties of the residues being repositioned (i.e. Trp or Tyr versus Phe). Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.

Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS

“…Communication of structural changes from extracellular and periplasmic detector domains to transmembrane helices that rely on the presence of a conserved proline residue at the top of the helix or in the loop joining two consecutive transmembrane helices is seen in numerous receptor structures, such as in the family of pentameric ligand-gated ion channels (e.g., the acetylcholine and gamma-aminobutyric acid [GABA] receptors in humans), where such changes are key parts of the gating mechanism (reviewed in reference 49 ). The resulting small displacements in the transmembrane helices of the EvgS dimer could activate the cytoplasmic HK activity of EvgS in a way analogous to those reported in a related family of sensory kinases that includes EnvZ ( 50 , 51 ).…”

Transmembrane Signal Transduction in Two‐Component Systems: Piston, Scissoring, or Helical Rotation?

“…The piston model originated from the analysis of chemoreceptor Tar sensor domain in apo and holo forms and was elegantly illustrated using cysteine cross‐linking and repositioning of helix‐flanking aminoacids . The model has also found support in simulations .…”

In vivocross-linking and transmembrane helix dynamics support a bidirectional non-piston model of signaling withinE. coliEnvZ

“…The piston-type model of transmembrane communication is founded upon the central tenant that the vertical position of TM2 relative to the lipid bilayer changes upon stimulus perception. Previous work with TM2 of Tar demonstrates that repositioning the aromatic residues at the cytoplasmic end of TM2, known as aromatic tuning (33), repositions the helix within a biological membrane (34) and that this repositioning causes an incremental change in signal output (33). These results served as an experimental framework to optimise SIDEKICK software capable of high-throughput parallelised coarse-grained molecular dynamics (CG-MD) simulations, which demonstrated that aromatic tuning repositioned the TM2 helix in silico in a manner consistent with both the in vivo results and a piston-type mechanism of transmembrane communication (35).…”

“…Various experimentation has also been performed with the aromatically tuned variants of TM2 from both Tar and EnvZ. Previously, a linear correlation was observed between the position of the aromatic residue in Tar TM2, the position of the helices in vitro and in silico and the signal output from each Tar receptor (33, 34, 40, 50). Here, in silico analysis of EnvZ TM2 demonstrates that such a linear correlation is absent and that EnvZ functions by a nonpiston mechanism in which both tilting and azimuthal rotation play a substantial role in modulation of signal output (Figure 5).…”