“The hydration level of a transmembrane hydrophilic motif modulates DesK activity”

CYBULKSKI, LE; MARTÍN, M; MANSILLA, MC; FERNÁNDEZ, A; DE MENDOZA, D

Tucumán, Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

SAIB

The histidine kinase DesK from Bacillus subtilis is a paradigmaticexample of a sensor specifically tailored to remodel membranefluidity when the temperature drops below ~30°C. In this work, weaddress the process by which DesK transmembrane segments(TMS) transmit temperature signals across the membrane byengineering the 5 TMS domain of the DesK into a single-TMSchimeric sensor. This so-called Minimal Sensor (MS) fully retainsin vivo and in vitro the sensing input and transmission output of theparental system. Progressive deletions of TM segments revealedthat only the first TM segment (TM1) is essential to regulate thekinase activity. Therefore, our engineered MS combines the Nterminal17-residue portion of TM1 with the C-terminal 14-residueportion of TM5 which is naturally fused to the cytosolic catalyticdomain. The MS N-terminus contains three hydrophilicaminoacids near the lipid-water interface creating an instability hotspot. This boundary-sensitive motif controls the sensing andtransmission activity. Accordingly, we hypothesize that membranethickness is the temperature agent that determines the signalingstate of the cold sensor by dictating the hydration level of the metastablehydrophilic spot. This conjecture is supported through thestudy of the signaling behavior of MS variants purposelyconstructed.