STRUCTURAL INSIGHTS INTO THE SIGNAL TRANSDUCTION MECHANISM OF THE COLD SENSOR DesK

ALBANESI, DANIELA; MARTÍN, MARIANA; TRAJTENBERG, FELIPE; MANSILLA, MARÍA CECILIA; ALZARI, PEDRO; BUSCHIAZZO, ALEJANDRO; DE MENDOZA, DIEGO

Rosario, Argentina

Workshop; 3er WORKSHOP IN CRYOBIOLOGY APPLIED TO MEDICAL SCIENCES; 2009

Centro Binacional de Criobiología Clínica y Aplicada, UNESCO Chair in Criobiology, Facultad de Ciencias Bioquímicas y Farmacéuticas Universidad Nacional de Rosario

Temperature sensing and adaptation are essential for the survival of living cells. Organisms respond to changes in environmental temperature either by regulating body temperature or through adaptive behavioral thermoregulation. Although great strides have been taken towards understanding the fundamental process of temperature detection in many organisms ranging from bacteria to man, the molecular mechanisms of thermosensing are yet to be discovered in most systems. The best documented early effect of environmental cold on cellular processes is a decrease in membrane fluidity. In bacteria, generally lacking cholesterol, membrane fluidity is largely determined by the proportion of unsaturated fatty acids in the phospholipids. A simplified way to visualize the problem is to consider the shape of the fatty acids. The straight chain saturated fatty acids are linear and pack efficiently to produce a bilayer that has a high phase  transition and low permeability properties. The double bond of unsaturated fatty acids introduces a pronounced kink in the chain, which disrupts the order of the bilayer and results in lower transition temperatures and higher permeability. Accumulating evidence indicates that cold induced membrane lipid rigidification is sensed by membrane proteins acting as cold sensor regulators. In bacteria, the most studied system leading to thermal adaptation is the Des pathway of Bacillus subtilis. This pathway is composed of the DesK/DesR two-component system (TCS) and Δ5-acyl desaturase (Δ5-Des), the unique desaturase in this organism encoded by the des gene. DesK is a prokaryotic histidine kinase that has an N-terminal sensor domain (~150 residues) composed of four or five transmembrane (TM) segments connected to a C-terminal cytoplasmic catalytic core (DesKC, ~220 residues). DesR is a cytoplasmic response regulator that specifically controls the expression of the des gene. DesK is the founding example of a membrane bound thermosensor suited to remodel membrane fluidity when the ambient temperature drops below ∼30ºC. Induction of the Des pathway is brought about by the ability of DesK to assume different signaling states in response to variations in membrane fluidity. At cold temperatures, the increase in the proportion of ordered membrane lipids favors a kinase-dominant state of DesK, which undergoes autophosphorylation in a conserved histidine residue(H188). The phosphorylated kinase then transfers the phosphate to DesR. DesR-P tetramer binds to the des promoter, leading to recruitment of RNA polymerase and activation of des transcription. Activation of des results in the synthesis of Δ5-Des, which introduces double bonds in the acyl chains of membrane lipids. These newly synthesized unsaturated fatty acids decrease the phase transition temperature of the phospholipids, favoring the phosphatase activity of DesK on DesR-P and turning off of transcription. Genetic andbiochemical evidence suggests that the balance of two antagonistic DesK activities determines the DesR phosphorylation state: a phosphate donor for DesR and a phosphatase activity for DesR-P. As the activity of the transcriptional activator DesR is modulated by its phosphorylation state, the output of the DesK/DesR signal transduction pathway is determined by switches between kinase-biased and phosphatase-biased DesK activities. The balance between these activities would be regulated by changes in growth temperature that, in turn, dictates the fluidity of membrane lipids. It has been shown that in vitro, DesKC catalyzes its autophosphorylation, the phosphorylation of DesR and the desphosphorylation of phospho-DesR. However, the switch of DesKC from a kinase-biased to a phosphatase-biased activity is not temperature-regulated in vivo, suggesting that DesK TM segments play a crucial role in thermosensing and thermoadaptation. To establish how fluctuations in ambient temperature affect the phosphorylation state of DesK, we solved the crystal structure of its kinase cytoplasmic domain in three signaling states and determined the functional properties of the full length sensor in pure lipids vesicles. We propose a model in which the kinase-on to kinase-off transition involves a complex rearrangement of the central coiled-coil four helix bundle domain controlled by the TM sensing domain, which in turn detects a thermal input stimulus through changes in the order of the lipid bilayer. This regulation mechanism could be operational in a wide range of sensor proteins detecting a variety of different stimuli.