PROLINE RESIDUES OF DESK AS TRANSMISSION ELEMENTS OF CONFORMATIONAL CHANGES DURING COLD-SHOCK RESPONSE

LUCIA PORRINI, DIEGO DE MENDOZA, MARIA C MANSILLA

Rosario

Congreso; IX CONGRESO ARGENTINO DE MICROBIOLOGIA GENERAL SAMIGE 2013; 2013

Bacillus subtilis responds to a sudden decrease in temperature by transiently inducing the expression of the des gene encoding for a lipid desaturase, D5-Des, which introduces a double bond into the acyl chain of preexisting membrane phospholipids. This D5-Des-mediated membrane remodeling is controlled by the cold-sensor DesK. The molecular detail of how the input signal is sensed by the transmembrane segments (TMS) of DesK and transmitted to the cytoplasmic catalytic domain is completely unknown. In order to answer this fundamental issue, we decided to identify residues critical for cold sensing. We found five proline residues in TMS of DesK which are conserved in several membrane-bound thermosensors. To investigate whether these prolines play an essential role in the sensing and transmission of cold stimulus, we mutated each residue individually to alanine. We first assayed the effect of Pro to Ala substitution on expression of the des gene using strain DAK3. This strain carries a DesK null mutation, expresses desR from Pxyl promoter and contains a Pdes-lacZ transcriptional fusion. This strain was transformed individually with plasmids expressing DesKPAs alleles. The b-galactosidase activity of these strains showed that all DesKPA mutants were unable to activate des transcription upon a temperature downshift. This could be due either to DesKPA mutants were locked in a phosphatase-dominant state or the mutations producing an inactive enzyme in both kinase and phosphatase activities. In order to answer this question we assayed in vivo phosphatase activity of DesKPA variants. Our findings show that DesK mutants retain the phosphatase activity, indicating that Pro to Ala replacement does not completely inactivate DesK but brings it to adopt a phosphatase dominant state. In an attempt to elucidate the mechanistic basis of this unexpected role of prolines on the signaling state of DesK we plan to isolate mutants of DesKPA that revert its phenotype deficient in kinase activity. The search for new mutants, now able to detect the order of membrane lipids, was performed by generating random mutations within the gene sequence coding for the TMSs (desKPAtms) by error prone PCR mutagenesis. Surprisingly we isolated two clones that incorporate a leucine to proline substitutions at position 174, which restored wild type phenotype in desKPA alleles. These new DesKPA-L174P mutants are able to response to changes in temperature and membrane composition. These results suggest that the helix distortion generated by Pro174 would be favoring the kinase conformation lost in DesKPA variants. Our findings show that the prolines in DesK TMS are critical for conformational changes that lead DesK to adopt a kinase state and so these residues could play an important role in signal transduction probably inducing regions of dynamic flexibility (hinges) in the helices in such a way as to bring cytoplasmic domains into optimal positions for catalysis.