STRUCTURAL ELEMENTS LEADING TO MODULATION OF THE ACTIVITIES OF DESK, A THERMOSENSOR FROM Bacillus subtilis

SAITA, EMILIO; TSAI, YI TING; DE MENDOZA, DIEGO; ALBANESI, DANIELA

Rosario

Congreso; IX Congreso de Microbiología General; 2013

Sociedad Argentina de Microbiología General

Temperature sensing and adaptation are essential for the survival of living cells. The best documented early effect of environmental cold on cellular processes is a decrease in membrane fluidity. In bacteria,the most studied system leading to thermal adaptation is the Des pathway of Bacillus subtilis. This pathway is composed of the DesK/DesR two-component system (TCS) and a D5-acyl desaturase (D5-Des), encoded by the des gene. Induction of the Des pathway is brought about by the ability of the histidine kinase DesK to assume different signaling states in response to variations in membrane fluidity. An increase in the proportion of ordered membrane lipids favors a kinase-dominant state of DesK, which undergoes autophosphorylation and then transfers the phosphate to the response regulator DesR. DesR-P binds to the des promoter inducing des transcription. Once synthesized, D5-Des introduces double bonds in the acyl chains of membrane lipids decreasing the transition temperature of the phospholipids. A more fluid membrane favors the phosphatase activity of DesK on DesR-P turning off des transcription. DesK is a prokaryotic histidine kinase that has an N-terminal sensor domain (~150 residues) composed of five transmembrane (TM) segments connected to a C-terminal cytoplasmic catalytic core (DesKC, ~220 residues). It represents the founding example of a membrane bound thermosensor suited to remodel membrane fluidity when the ambient temperature drops below ~30°C. We have recently reported six crystal structures of the entire cytoplasmic catalytic core of DesK trapped in three conformational states corresponding to alternate functions of the protein along its catalytic cycle, allowing us to propose a model to account for the regulation mechanism of the catalytic activities of this protein. Comparison of the different structures invited the hypothesis that contacts between the central dimerization histidine phosphotransfer domain (DHp) and the ATP-binding domains (ABDs), as well as a dynamic N-terminal parallel coiled-coil, support a labile association to be released for autophosphorylation and maintained for the phosphatase activity, under control of the sensor domain upon signal perception. To test this hypothesis we have performed structure-based mutagenesis in order to stabilize and destabilize the inter-domain interactions. A set of B. subtilis strains were generated for in vivo analysis and a purification protocol was also set up to obtain some of the target proteins in their active form for in vitro studies. The results obtained from in vivo and in vitro activity assays highlight the relevance of these structural elements to control the output activity of DesK and confirm the importance of the rotational and shifting movements in the conserved DHp domain on the signal transduction mechanism of this sensor protein.