Driving the catalytic activity of a transmembrane thermosensor kinase.

DesK is a Bacillus thermosensor kinase that is inactive at high temperatures but turns activated when the temperature drops below 25 °C. Surprisingly, the catalytic domain (DesKC) lacking the transmembrane region is more active at higher temperature, showing an inverted regulation regarding DesK. How does the transmembrane region control the catalytic domain, repressing activity at high temperatures, but allowing activation at lower temperatures? By designing a set of temperature minimized sensors that share the same catalytic cytoplasmic domain but differ in number and position of hydrogen-bond (H-bond) forming residues along the transmembrane helix, we are able to tune, invert or disconnect activity from the input signal. By favoring differential H-bond networks, the activation peak could be moved towards lower or higher temperatures. This principle may be involved in regulation of other sensors as environmental physicochemical changes or mutations that modify the transmembrane H-bond pattern can tilt the equilibrium favoring alternative conformations.

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

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.

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

The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially "buoy" to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we identify a "linker region" (KSRKERERLEEK) that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/phosphatase activity ratio, as required to modulate the DesK output response.

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

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.

A Transmembrane Histidine Kinase Functions as a pH Sensor.

The two-component system DesK-DesR regulates the synthesis of unsaturated fatty acids in the soil bacteria Bacillus subtilis. This system is activated at low temperature and maintains membrane lipid fluidity upon temperature variations. Here, we found that DesK-the transmembrane histidine kinase-also responds to pH and studied the mechanism of pH sensing. We propose that a helix linking the transmembrane region with the cytoplasmic catalytic domain is involved in pH sensing. This helix contains several glutamate, lysine, and arginine residues At neutral pH, the linker forms an alpha helix that is stabilized by hydrogen bonds in the i, i + 4 register and thus favors the kinase state. At low pH, protonation of glutamate residues breaks salt bridges, which results in helix destabilization and interruption of signaling. This mechanism inhibits unsaturated fatty acid synthesis and rigidifies the membrane when Bacillus grows in acidic conditions.