Mechanism of signal detection and signal transduction in the thermosensor DesK

VAZQUEZ DANIELA; INDA, MARÍA EUGENIA;; DE MENDOZA DIEGO; CYBULSKI LARISA ESTEFANÍA

Rosario-Santa Fe

Congreso; Congreso 50 años de la Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular; 2014

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

The Des circuit in B. subtilis is designed to adjust the composition of the membrane lipids in response to the growth temperature, regulating the expression of an acyl-lipid desaturase. This circuit is controlled by the DesKR two- component system. DesK is a transmembrane protein with five segments (TMS) which has kinase and phosphatase activities at 25 or 37° respectively. What biophysical feature gives DesK the ability to act as a sensor? Recently we designed a chimerical protein having a single TMS, which works like full length DesK, and was called minimal sensor. This discovery allowed us to study a complex phenomenon using a simple model. We found a serine zipper motif at the C-terminus of the TMS, located at one face of the helix. The zipper tends to form inter-helical hydrogen bonds at low temperatures, when the membrane is thicker and dehydrated. To test our model of activation, which implies bringing two TM helices closer to stabilize a dimer interface, mediated by the serine zipper, we replaced zipper serines by residues with strong or negligible capacity to form hydrogen bond, using the method of Quick change. Variants were cloned into the expression plasmid pHPKS, which were used to transform Bacillus subtilis and then measure b-galactosidase activity at 25ºC or 37°C. We found that residues with hydrogen bond capacity stabilize the active DesK conformation.