Design and mechanism of a minimal sensor that responds to membrane fluidity

CYBULSKI, LARISA ESTEFANÍA; FERNANDEZ, ARIEL; DE MENDOZA, DIEGO

Montevideo, Uruguay

Conferencia; 6 International Conference of Biological Physics- ICBP; 2007

ICBP- International Union of Pure and Applied Physics, Federación Iberoamericana de Sociedades de Física, Southern Cone Biophysical Societies, Asociación Física Argentina,Sociedad Argentina de Biofísica, Sección Biofísica de la Sociedad Uruguaya de Bioci

Bacteria, fungi, and plants have developed two component systems (TCS) to deal with the variable environmental conditions. They are composed of a histidin-kinase (HK), the sensor protein that switches between two opposite activities (kinase and phosphatase) in response to a given signal. The other component is a response regulator (RR), effector protein that becomes phosphorylated by the HK, and whose DNA_ binding properties vary dramatically upon phosphorylation. The B. subtilis DesKR TCS has been identified as the first system that senses changes in membrane lipid fluidity and responds accordingly. It is composed of a membrane HK, DesK, a cytoplasmic RR, DesR, and the effector enzyme, D5-desaturase. Under conditions of restricted membrane fluidity DesK phosphorylates DesR, enabling its binding to the desaturase promoter. A tetramer of phosphorylated DesR positioned on Pdes recruits the RNA Polymerase to increase the transcription of the acyl-lipid desaturase. This enzyme introduces double bonds into the acyl chains of membrane lipids. The newly synthesized unsaturated fatty acids decrease the phase transition temperature of the phospholipids, favoring the phosphatase activity of DesK, what results in the interruption of des transcription. Several membrane topology prediction programs applied to the protein sequence of DesK predict four primary transmembrane segments (TMS) and one putative secondary TMS. We analyzed the topology of DesK and found that DesK has in fact five TMS instead of four. Sequential deletion of different TMS led us to the conclusion that i) TMS are responsible not only for anchoring the cytoplasmic kinase/phosphatase domain of DesK to the membrane, but also to perceive the signal, and ii) the first amphipathic helix of DesK is a non predicted TMS that is crucial for the detection and signaling of the membrane fluidity in B. subtilis, since its remotion leads to constitutive kinase activity regardless of the growth temperature. To obtain a simple system that could enable us to study the sensing mechanism of Desk we constructed a series of fusion proteins  composed by the cytoplasmic domain of DesK and one of several chimerical TMSs envisioned as possessing the critical elements involved in sensing. One of these fusion proteins, with a unique chimerical TMS behaves as DesK wild type, so we called it DesK-Minimal Sensor (DesK-MS), due to its simplicity. A series of site-directed mutants located in this chimerical TMS are being constructed to understand the mechanism of fluidity sensing.