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.

Importance: The 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.

FIG 1

Cold detection in B. subtilis. (A) The histidine kinase DesK (five TM-spanning segments) and the synthetic MS-DesK protein localize at the lipid bilayer. At low temperature (25°C), the level of order in the structures of the acyl chains of lipids increases, promoting a kinase-active state. DesK autophosphorylates and transfers the phosphoryl group to Asp54 of the DesR dimeric response regulator (two gray circles), inducing a conformational change that results in exposure of the helix-turn-helix (HTH)–DNA-binding domain. Two dimers of phosphorylated DesR bind two adjacent, nonidentical binding sites within the des promoter (40), leading to recruitment of the RNA polymerase to activate expression of the desaturase gene. (B) At higher temperature (37°C), the disordered lipids favor the phosphatase-active state of DesK, leading to dephosphorylation of phospho-DesR; thus, des transcription is turned off. The dyad Phe8-Lys10 and the serine zipper are highlighted in the insets to show their locations at the water-lipid interphase. (C) Sequence of the functional synthetic TMS of minimal sensor DesK and the nonfunctional TM1 and TM5. (D) DesK and MS kinase activities at 25°C and 37°C. MU, Miller units. (Adapted from reference with permission of the publisher.)

FIG 2

Bacillus membranes are thicker at lower temperature. Bacillus cells were grown at 37°C to an OD of 1, and lipids were extracted using the method of Bligh and Dyer (18). Liposomes prepared with these lipids were used to determine the total hydrophobic thickness (inset) across the bilayer by modeling the small-angle X-ray scattering raw data in the main graph as a function of temperature. The subtle shift of the curves upon heating indicates the change of the thickness of the membrane. The associated coefficient for thermal expansion perpendicular to the membrane (α_⊥) was found to be −3.1 × 10⁻³ K⁻¹, a value expected for bilayer membranes (13). The negative sign reflects the thinning of the membrane that occurs upon heating. Hydrophobic thickness changes about 1 Å over the physiological range studied (25 to 37°C). a.u., arbitrary units.

FIG 3

MS-DesK reconstituted in liposomes displays autokinase and phosphatase activities. (A) The autokinase activity of detergent-dissolved MS-DesK is not regulated by temperature. MS-DesK solubilized in 0.5% Brij 58 was incubated with [γ-³²P]ATP at either 25 or 37°C, and the autokinase activity was analyzed at different interval points by SDS-PAGE followed by autoradiography. (B) DesK is integrated into liposomes. Proteoliposomes were purified by a sucrose gradient centrifugation in the absence (–) or presence (+) of 0.5 M KCl and later analyzed by Western blotting using anti-His antibodies. (C) MS-DesK integrated into liposomes was incubated with [γ-³²P]ATP at either 25 or 37°C, and the autokinase activity was measured by taking samples at different time points. The autokinase activity of proteoliposomes is regulated by temperature, showing that MS-DesK must be integrated into a lipid bilayer to be thermoregulated. (D) Quantification of the MS-DesK kinase activity shown in panel C by the use of Image Quant software (version 5.2). AU, arbitrary units. (E) MS-DesK phosphatase activity. Proteoliposomes containing MS-DesKC were incubated at 37°C with purified DesR-P. The dephosphorylation reactions were analyzed by SDS-PAGE followed by autoradiography. (F) The total amount of DesR-P (expressed in arbitrary units) present in each well was determined by densitometry as described for panel D; the total labeling of DesR-P at the beginning of the reaction (0 min) was considered 100%. The graph shows the percentage of DesR-P protein (prot) remaining as a function of time.

FIG 4

Activity of MS-DesK mutants. (A) Sequences of the N terminus of MS-DesK variants, with point mutations that eliminate (K10L) or duplicate (L11K) the positive charge. WT, wild type. (B) Cells expressing MS-DesK variants were grown at 37°C and transferred to 25°C at an OD at 525 nm (OD₅₂₅) of 0.3. β-Galactosidase activity was assayed every hour in independent triplicates. The data shown are expressed as averages of the results from three independent experiments and correspond to 4 h after the cold shock; error bars represent the standard deviations for each experimental repetition (10). (C) Proteoliposomes containing MS-DesK or its variants were incubated with [γ-³²P]ATP at 25°C, and the autokinase activity was determined using an ADP-Glo kinase assay. The total amount of each MS-DesK variant was determined by densitometry; equal amounts of protein were used for each reaction. (D) The corresponding relative initial velocities were calculated from the slopes of the curves shown in panel C and compared, considering MS-DesK as 100%. (E and F) The phosphatase activity of MS-DesK variants was determined as described for Fig. 3E. (G) Relative initial velocities were calculated from the slope of the curves shown in panel F, taking the activity of MS-DesK as 100%.

FIG 5

The dyad F8-K10 is critical for determining the signaling state of MS-DesK. Different MS-DesK variants with mutations in the dyad F8-K10 were analyzed for in vivo activity. The conservative replacement K10R does not eliminate thermoregulation of MS-DesK activity; however, the replacement of K10L and K10E abolished high kinase activity at low temperatures, suggesting that a positive charge is required at position 10. The conservative replacement F8L also maintains regulation, as expected, because leucine and phenylalanine have the same hydrophobic profile. F8A behaves much like K10E, probably because of the loss of hydrophobicity at residue 8. F8W is constitutively active due to the strong preference of Trp for the water-lipid interphase. The double mutant F8A-K10L lacks kinase activity, since both components of the thermosensitive dyad are eliminated. The double mutant F8A-L11K is also constitutively active; since the presence of an extra lysine residue located deeper (1.5 Å) inside the membrane exerts an outward pull on the TMS even at higher temperatures, when the membrane is thinner. β-Galactosidase activity was determined as described for Fig. 4B.