An Analysis of the Solution Structure and Signaling Mechanism of LovK, a Sensor Histidine Kinase Integrating Light and Redox Signals

摘要

Flavin-binding LOV domains are broadly conserved in plants, fungi, archaea, and bacteria.nThese ≈100-residue photosensory modules are generally encoded within larger, multidomain proteins thatncontrol a range of blue light-dependent physiologies. The bacterium Caulobacter crescentus encodes a solublenLOV-histidine kinase, LovK, that regulates the adhesive properties of the cell. Full-length LovK is dimeric asnare a series of systematically truncated LovKconstructs containing only theN-terminal LOV sensory domain.nNonconserved sequence flanking the LOV domain functions to tune the signaling lifetime of the protein. Sizenexclusion chromatography and small-angle X-ray scattering (SAXS) demonstrate that the LOV sensorndomain does not undergo a large conformational change in response to photon absorption. However, limitednproteolysis identifies a sequence flanking the C-terminus of the LOV domain as a site of light-induced changenin protein conformation and dynamics. On the basis of SAXS envelope reconstruction and bioinformaticnprediction, we propose this dynamic region of structure is an extended C-terminal coiled coil that links thenLOV domain to the histidine kinase domain. To test the hypothesis that LOV domain signaling is affected byncellular redox state in addition to light, we measured the reduction potential of the LovK FMN cofactor. Thenmeasured potential of -258 mV is congruent with the redox potential of Gram-negative cytoplasm duringnlogarithmic growth (-260 to -280 mV). Thus, a fraction of LovK in the cytosol may be in the reduced statenunder typical growth conditions. Chemical reduction of the FMN cofactor of LovK attenuates the light-ndependent ATPase activity of the protein in vitro, demonstrating that LovK can function as a conditionalnphotosensor that is regulated by the oxidative state of the cellular environment.