The hydration level of a transmembrane hydrophilic motif modulates DesK activity

CYBULSKI, LARISA E.; MARTIN MARIANA, MANSILLA MC, FERNÁNDEZ ARIEL, DE MENDOZA, DIEGO

Tucumán, Argentina

Congreso; - XLV Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB).; 2009

The histidine kinase DesK from Bacillus subtilis is a paradigmatic example of a sensor specifically tailored to remodel membrane fluidity when the temperature drops below ~30°C. In this work, we address the process by which DesK transmembrane segments (TMS) transmit temperature signals across the membrane by engineering the 5 TMS domain of the DesK into a single-TMS chimeric sensor. This so-called Minimal Sensor (MS) fully retains in vivo and in vitro the sensing input and transmission output of the parental system.  Progressive deletions of TM segments revealed that only the first TM segment (TM1) is essential to regulate the kinase activity.  Therefore, our engineered MS combines the N-terminal 17-residue portion of TM1 with the C-terminal 14-residue portion of TM5 which is naturally fused to the cytosolic catalytic domain. The MS N-terminus contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. This boundary-sensitive motif controls the sensing and transmission activity. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. This conjecture is supported through the study of the signaling behavior of MS variants purposely constructed.