A lipid-mediated conformational switch modulates the thermosensing activity of DesK

Inda, María Eugenia; Vandenbranden, Michel; Fernández, Ariel; Mendoza, Diego de; Ruysschaert, Jean Marie; Cybulski, Larisa E.

doi:10.1073/pnas.1317147111

Cited by 73 publications

(70 citation statements)

References 23 publications

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“…Serine residues located at the C terminus of the TMS penetrate deeper into the hydrophobic core of the membrane, which results in dehydration and repositioning of these residues to establish H-bonds with homologous serine residues on the other monomer (12). The pulling force of K10 triggered by the thickening of the membrane at low temperatures promotes the incorporation of an extra helical turn at the C terminus of the TMS (11). We propose that this reorganization involves a rotation of both the TMS and the contiguous cytoplasmic coiled-coil, which in turn results in a kinase-competent state (36).…”

Section: Figmentioning

confidence: 99%

“…It has been proposed that a lysine residue (K10) located near the N terminus of the TMS and a serine zipper located at the C terminus of the TMS, which is connected to the linker region at the intracellular membrane-water interface, act together as a molecular caliper to detect changes in membrane thickness that occur as a consequence of temperature variations ( Fig. 1) (10)(11)(12). It has been reported that the thickness of lipid bilayers formed by pure synthetic phospholipids increases when temperature decreases (13,14).…”

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The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper

et al. 2016

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Thermosensors detect temperature changes and trigger cellular responses crucial for survival at different temperatures. The thermosensor DesK is a transmembrane (TM) histidine kinase which detects a decrease in temperature through its TM segments (TMS). Here, we address a key issue: how a physical stimulus such as temperature can be converted into a cellular response. We show that the thickness of Bacillus lipid membranes varies with temperature and that such variations can be detected by DesK with great precision. On the basis of genetic studies and measurements of in vitro activity of a DesK construct with a single TMS (minimal sensor DesK [MS-DesK]), reconstituted in liposomes, we propose an interplay mechanism directed by a conserved dyad, phenylalanine 8-lysine 10. This dyad is critical to anchor the only transmembrane segment of the MS-DesK construct to the extracellular water-lipid interphase and is required for the transmembrane segment of MS-DesK to function as a caliper for precise measurement of membrane thickness. The data suggest that positively charged lysine 10, which is located in the hydrophobic core of the membrane but is close to the water-lipid interface, pulls the transmembrane region toward the water phase to localize its charge at the interface. Nevertheless, the hydrophobic residue phenylalanine 8, located at the N-terminal extreme of the TMS, has a strong tendency to remain in the lipid phase, impairing access of lysine 10 to the water phase. The outcome of this interplay is a fine-tuned sensitivity to membrane thickness that elicits conformational changes that favor different signaling states of the protein. IMPORTANCEThe ability to sense and respond to extracellular signals is essential for cell survival. One example is the cellular response to temperature variation. How do cells "sense" temperature changes? It has been proposed that the bacterial thermosensor DesK acts as a molecular caliper measuring membrane thickness variations that would occur as a consequence of temperature changes and activates a pathway to restore membrane fluidity at low temperature. Here, we demonstrated that membrane thickness variations do occur at physiological temperatures by directly measuring Bacillus lipid membrane thickness. We also dissected the N-terminal sensing motif of MS-DesK at the molecular-biophysical level and found that the dyad phenylalanine-lysine at the water-lipid phase is critical for achievement of a fine-tuned sensitivity to temperature. T he ability to sense and respond to subtle variations in environmental temperature is critical for all kingdoms of life. Temperature changes modify protein activity directly but also indirectly, by modifying biophysical properties of membrane lipids, thereby affecting the activity of membrane-embedded or membrane-associated proteins and threatening the viability of the cell (1-3).DesK is a transmembrane (TM) histidine kinase which, together with its cognate response regulator, DesR, operates in Bacillus subtilis to maintain membrane fluidity...

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The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper

et al. 2016

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“…For this reason, this has been called the sunken-buoy (SB) motif (5). In addition to the SB motif, a charged linker region at the intracellular membrane-water interface was found to be important for activity (10). It has been proposed that both motifs act together as a molecular gauge that senses membrane thickness and thereby regulates the switching of activity of the intracellular catalytic domain of DesK (10).…”

Section: Significancementioning

confidence: 99%

“…In addition to the SB motif, a charged linker region at the intracellular membrane-water interface was found to be important for activity (10). It has been proposed that both motifs act together as a molecular gauge that senses membrane thickness and thereby regulates the switching of activity of the intracellular catalytic domain of DesK (10). The molecular details of the mode of action of this molecular gauge have remained elusive, however.…”

Section: Significancementioning

confidence: 99%

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness

Cybulski¹,

Ballering

Moussatova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DesK is a bacterial thermosensor protein involved in maintaining membrane fluidity in response to changes in environmental temperature. Most likely, the protein is activated by changes in membrane thickness, but the molecular mechanism of sensing and signaling is still poorly understood. Here we aimed to elucidate the mode of action of DesK by studying the so-called "minimal sensor DesK" (MS-DesK), in which sensing and signaling are captured in a single transmembrane segment. This simplified version of the sensor allows investigation of membrane thickness-dependent protein-lipid interactions simply by using synthetic peptides, corresponding to the membrane-spanning parts of functional and nonfunctional mutants of MS-DesK incorporated in lipid bilayers with varying thicknesses. The lipid-dependent behavior of the peptides was investigated by circular dichroism, tryptophan fluorescence, and molecular modeling. These experiments were complemented with in vivo functional studies on MS-DesK mutants. Based on the results, we constructed a model that suggests a new mechanism for sensing in which the protein is present as a dimer and responds to an increase in bilayer thickness by membrane incorporation of a C-terminal hydrophilic motif. This results in exposure of three serines on the same side of the transmembrane helices of MS-DesK, triggering a switching of the dimerization interface to allow the formation of a serine zipper. The final result is activation of the kinase state of MS-DesK.thermosensing | two-component system | lipid-protein interaction | helix-helix interaction | transmembrane helix dimerization

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“…We assume that the linker in CadC transduces the signal and defines the orientation of the DNA-binding domain. Likewise a linker region in DesK, a multipass transmembrane histidine kinase of Bacillus subtilis, mediates signal transduction by adopting two conformational states[31]. The histidine kinase EnvZ also has a cytoplasmic linker of approximately 50 amino acids.…”

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Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli

Buchner

Schlundt

Lassak

et al. 2015

Journal of Molecular Biology

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The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH<6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain.

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A lipid-mediated conformational switch modulates the thermosensing activity of DesK

Cited by 73 publications

References 23 publications

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper

The Single Transmembrane Segment of Minimal Sensor DesK Senses Temperature via a Membrane-Thickness Caliper

Activation of the bacterial thermosensor DesK involves a serine zipper dimerization motif that is modulated by bilayer thickness

Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli

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