Comprehensive Insights into the Bacillus subtilis Des Pathway: Visualizing Components by Single-Molecule Super-Resolution Microscopy and Advancing Structural Studies with Anti-DesK Nanobodies

JULIÁN PEREYRA; VANINA ALZOGARAY; GONZALO ESCALANTE; LUCÍA LÓPEZ; MECHALY ARIEL; FERNANDO STEFANI; DANIELA ALBANESI

Rosario

Congreso; Sociedad Argentina de Investigaciones en Bioquímica 2023; 2023

SAIB

The Des signaling pathway of Bacillus subtilis represents a paradigm for studying sensors that regulate membrane fluidity in both unicellular and multicellular eukaryotic organisms. This pathway comprises an integral membrane desaturase, Δ5-Des, encoded by the des gene, and the two-component system DesK/DesR, which controls its expression. DesK is a transmembrane multifunctional histidine kinase with autokinase, phosphotransferase, and phosphatase activities. When the temperature drops below 30ºC, accompanied by a decrease in membrane fluidity, DesK acts as a kinase and autophosphorylates. The phosphoryl group is then transferred to DesR. Phosphorylated DesR (DesR-P) activates des expression. Once synthesized, Δ5-Des desaturates the acyl chains of the membrane phospholipids, restoring their fluidity. Upon an increase in membrane fluidity, DesK adopts its phosphatase state, dephosphorylating DesR-P to turn off the response. Structural biology, biochemical and molecular biology studies with the soluble cytoplasmic domain of DesK (DesKC) allowed us to elucidate the molecular mechanism for the catalytic regulation of this sensor protein. However, the mechanism of signal detection and transmission by DesK´s transmembrane segments remains a mystery. Moreover, at present, there is limited information about how signaling pathways, such as the Des pathway, are regulated within the cell in terms of the localization and dynamics of the proteins involved in the adaptive response. In vitro studies have revealed differential interactions between DesK and DesR depending on the functional status of DesK. Additionally, it has been determined that both exogenous and endogenous unsaturated fatty acids induce the phosphatase state of DesK, suggesting a potential interaction or co-localization with Δ5-Des to efficiently halt the adaptive response. These findings have led us to hypothesize that changes in membrane fluidity could impact the distribution and interaction of the components of the Des pathway within the cell. To test this hypothesis, we aim to precisely localize and co-localize the components of the Des pathway (DesK, DesR, Δ5-Des) using single-molecule super-resolution fluorescence microscopy techniques under various membrane fluidity conditions. To this end, we have developed specific anti-DesK nanobodies fused to a docking DNA oligomers by "click chemistry" or to an HA (Hemagglutinin) tag at the C-terminus that were successfully used, respectively, in DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) and STORM (Stochastic Optical Reconstruction Microscopy) super-resolution microscopy experiments. Moreover, we have initiated Cryo-EM (Cryo-Electron Microscopy) studies with the anti-DesK nanobodies-DesK complexes. This work is a first step towards understanding the functioning of the Des pathway from a structural and cellular point of view.