PROLINE TO THE RESCUE: A POINT MUTATION IN THE ESSENTIAL 2-HELIX COLIED COIL REVEALS MECHANISTIC DETAILS OF Bacillus subtilis HISTIDINE KINASE DesK

PILAR FERNÁNDEZ; DANIELA ALBANESI; LUCIA PORRINI; DIEGO DE MENDOZA; MARIA CECILIA MANSILLA

Buenos Aires

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

SAIB

Bacillus subtilisresponds to a sudden decrease in temperature by transiently inducing theexpression of the des gene encoding alipid desaturase, which introduces a double bond into the acyl chain ofpreexisting membrane phospholipids. This membrane remodeling is controlled bythe cold-sensor DesK, through the reversible formation of a two-helix coiled coil (2-HCC)in the fifth transmembrane (TM) segment and the N-terminus of thecytoplasmic domain. Byrandom mutagenesis, we isolated a point mutation in the 2-HCC (L174P) that could restore the cold sensing ability of single Proto Ala replacement mutants in TM1 and TM5. To understand how this modified2-HCC functions, we constructed a series of mutants by site directedmutagenesis, and expressed them in strain DAK3, engineered to test kinaseactivity by measuring β-galactosidase activity. As expected, when two Pro residueslocated in different TM segments were simultaneously replaced by Ala, or thezipper of serines of TM5 was disrupted, the protein showed only phosphataseactivity. However, when L174P mutation was incorporated, the thermosensing capacityof all the sensor mutant proteins was restored. So, we wondered if thismodified 2-HCC was indeed sensing temperature, independentlyof the membrane domain. However, when we incorporated L174P mutationinto a non-cold responding DesK allele, composed of TM5 linked to thecytoplasmic domain, we found that this modification was not sufficient to turnthis protein into a cold sensor, indicating that structural elements of thesensor domain are necessary for signal detection and transduction.Thepresent work supports previous analysis suggesting that the input signal mustpromote the rotation of the 2-HCC to destabilize it. We believe that introductionof Pro174 results in a slight destabilization of the 2-HCC that favors thekinase-competent state, counterbalancing modifications into the sensing domain.