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Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor.

ABSTRACT: The Escherichia coli Tsr protein contains a periplasmic serine-binding domain that transmits ligand occupancy information to a cytoplasmic kinase-control domain to regulate the cell's flagellar motors. The Tsr input and output domains communicate through conformational changes transmitted through a transmembrane helix (TM2), a five-residue control cable helix at the membrane-cytoplasm interface, and a four-helix HAMP bundle. Changes in serine occupancy are known to promote TM2 piston displacements in one subunit of the Tsr homodimer. We explored how such piston motions might be relayed through the control cable to reach the input AS1 helix of HAMP by constructing and characterizing mutant receptors that had one-residue insertions or deletions in the TM2-control cable segment of Tsr. TM2 deletions caused kinase-off output shifts; TM2 insertions caused kinase-on shifts. In contrast, control cable deletions caused kinase-on output, whereas insertions at the TM2-control cable junction caused kinase-off output. These findings rule out direct mechanical transmission of TM2 conformational changes to HAMP. Instead, we suggest that the Tsr control cable transmits input signals to HAMP by modulating the intensity of structural clashes between out-of-register TM2 and AS1 helices. Inward displacement of TM2 might alter the sidechain environment of control cable residues at the membrane core-headgroup interface, causing a break in the control cable helix to attenuate the register mismatch and enhance HAMP packing stability, leading to a kinase-off output response. This helix-clutch model offers a new perspective on the mechanism of transmembrane signaling in chemoreceptors.

SUBMITTER: Ames P

PROVIDER: S-EPMC5023463 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor.

Ames Peter P Hunter Samuel S Parkinson John S JS

Journal of molecular biology 20160324 19

The Escherichia coli Tsr protein contains a periplasmic serine-binding domain that transmits ligand occupancy information to a cytoplasmic kinase-control domain to regulate the cell's flagellar motors. The Tsr input and output domains communicate through conformational changes transmitted through a transmembrane helix (TM2), a five-residue control cable helix at the membrane-cytoplasm interface, and a four-helix HAMP bundle. Changes in serine occupancy are known to promote TM2 piston displacemen ...[more]

PMID: 27019297

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Project description:UnlabelledThe transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements.ImportanceThe Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane.

Project description:We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59]. We have further investigated this transition and find that the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterioopsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are 5.46 for residues 72-248 (Cys 163) and 7.36 for residues 1-166 (Lys 41). When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding in the range of -0.54 to -3.5 kcal/mol. The results suggest that the C-terminal helix of bacterioopsin is less stably folded than the N-terminal helices. We analyzed the pairwise helix-helix interaction surfaces of bacteriorhodopsin and three other seven-transmembrane-helix proteins on the basis of crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl-terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high-resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix interactions.

Dataset Information

Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor.

Publications

Evidence for a Helix-Clutch Mechanism of Transmembrane Signaling in a Bacterial Chemoreceptor.

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