Structural studies of the binary complex DesKC-DesR from B. subtilis, a thermal sensor two-component system”

TRAJTENBERG, F; ALBANESI, D; CYBULKSKI, LE; MANSILLA MC; OBAL, G; PRITSCH, O; DE MENDOZA D; BUSCHIAZZO, A

Montevideo, Uruguay

Congreso; 6th International Conference of Biological Phisics.; 2007

Bacteria have developed a quite diverse set of mechanisms for sensing and adapting to changes in their environment. In  B. subtilis, temperature variations regulate the expression of a trans-membrane desaturase, which is able to control the degree of membrane fluidity. The input signals in this temperature-sensing pathway are transduced by a  "two-components" system involving an ATP  dependent Histidine kinase (DesK) and a response   regulator (DesR) (Aguilar et al. 2001 EMBO  J, 20:1681). DesK has five transmembrane segments that define the sensor domain, and a   cytoplasmic C-terminal region (DesKC) that harbors the dimerization and  catalytic domains. DesR is a transcription factor, with an N-terminal phospho-receiving domain and a C-terminal  DNA-binding HTH motif. To understand how is the signal actually transmitted and why different homologous transduction pathways in the cell don´t cross-talk, we want to solve the crystal structures of DesKC in complex with specific ligands and, most importantly, the binary DesKC-DesR complex in different physiologically relevant conditions. We have expressed and purified recombinant  DesKC and DesR. We have been able to reproduce the crystals of DesKC alone that had been identified by our collaborators (Albanesi et al., unpublished results), and we have recently solved the crystal structure of DesKC bound to ATP, observing the ordering of the ATP-lid loop that wasn´t visible before. Further on, we started our work on the DesKC-DesR complex by analyzing their interaction in solution using size exclusion chromatography and surface plasmon resonance. Preliminary results confirm that, although quite sensitive to incubation conditions, DesKC and DesR do interact forming a stable binary complex. The quantitation of kinetic and equilibrium constants are in progress. We have set extensive assays to screen for conditions to crystallize the DesKC-DesR complex. Indeed, we have identified very promising clues that are currently being optimized. Once suitable crystals are obtained, we will proceed to single crystal diffraction studies in our lab, comparing the effects of different ligands and/or substrate derivatives, including phosphate intermediary-state analogs.