Proline to the Rescue: a Point Mutation in the Essential 2-Helix Coiled Coil Reveals Mechanistic Details of Bacillus subtilis Histidine Kinase DesK

PORRINI, LUCÍA; MANSILLA, MARÍA CECILIA; ALBANESI, DANIELA; DE MENDOZA, DIEGO; FERNÁNDEZ, PILAR

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias. LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2017

Sociedades de Biociencias. Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Bacillus subtilis responds to a sudden decrease in temperature by transiently inducing the expression of the des gene encoding a lipid desaturase, which introduces a double bond into the acyl chain of preexisting membrane phospholipids. This membrane remodeling is controlled by the cold-sensor DesK, through the reversible formation of a continuous two-helix coiled coil (2-HCC), including the fifth transmembrane (TM) segment and the N-terminus of the cytoplasmic domain. By random mutagenesis, we isolated a point mutation in the 2-HCC (L174P) that could restore the cold sensing ability of single Pro to Ala replacement mutants in TM1 and TM5. To understand how this modified 2-HCC functions, we constructed a series of mutants by site directed mutagenesis, and expressed them in strain DAK3, engineered to test kinase activity by measuring β-galactosidase activity. As expected, when two Pro residues located in different TM segments were simultaneously replaced by Ala, or the zipper of serines of TM5 was disrupted, the protein showed only phosphatase activity. However, when L174P mutation was incorporated, the thermosensing capacity of all the mutant variants of the sensor was restored. So, we wondered if this modified 2-HCC was indeed sensing temperature, independently of the membrane domain. However, when we incorporated L174P mutation into a non-cold responding DesK allele, composed of TM5 linked to the cytoplasmic domain, we found that this modification was not sufficient to turn this protein into a cold sensor, indicating that structural elements of the sensor domain are necessary for signal detection and transduction. The present work supports previous analysis suggesting that the input signal must promote the rotation of the 2-HCC to destabilize it. We believe that introduction of Pro174 results in a slight destabilization of the 2-HCC that favors the kinase-competent state, counterbalancing modifications into the sensing domain.