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

Abstract

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...

Microdomain formation is a general property of bacterial membrane proteins and induces heterogeneity of diffusion patterns

“…7b clearly show an increase in the diffusion coefficients upon rise in temperature, especially for proteins with smaller number of transmembrane domains. With a membrane thickness h of 2.7 nm [56, 57], we found a membrane viscosity of 1.16 ± 0.43Pa · s at 23 °C, 0.56 ± 0.06Pa · s at 37 °C, and 0.39 ± 0.04Pa · s at 43 °C in good agreement with values in the literature [58]. We also verified that the membrane viscosity decreases with the increasing temperature (inset of Fig.…”

Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase

“…The merged functional TMS was built by fusing 17 residues corresponding to the N-terminus of TM1 with 14 residues of the C-terminus of TM5 and the cytoplasmic catalytic domain of DesK (Figure 1A, right panel). This hybrid construction mimics the full-length protein: it is more active at 25 • C than at 37 • C. As the MS-DesKC lacks an important portion of the TM domain, its regulation is not as tight as that of the full length protein; nevertheless, it allowed us to study at molecular level the mechanism of thermosensing [12,13]. We have demonstrated that a group of Serine residues located towards the C-terminus of TM5 could work as a zipper to transmit the signal [14].…”

Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins

“…Maintenance of cell membrane homeostasis relies on regulatory proteins that sense and respond to properties such as lipid composition, thickness, compressibility, lateral mobility and curvature [ 1 , 2 ]. The best understood example is perhaps the bacterial multi-pass protein DesK that undergoes a conformational change in response to increased membrane rigidification, resulting in the activation of its kinase domain [ 3 – 6 ]. Similarly, the yeast single-pass plasma membrane protein Mga2 rotates along its long axis when the surrounding acyl chains are densely packed, also resulting in its activation [ 7 ].…”