Crystallization and initial X-ray diffraction analysis of the multi-domain Brucella blue light-activated histidine kinase LOV-HK in its illuminated state

JIMENA RINALDI; IGNACIO FERNÁNDEZ; LUCÍA M. POTH; WILLIAM E. SHEPARD; MARTIN SAVKO; FERNANDO A. GOLDBAUM; SEBASTIÁN KLINKE

Biochemistry and Biophysics Reports

Elsevier

Año: 2018 vol. 16 p. 39 - 43

2405-5808

The pathogenic bacterium Brucella abortus codes for a multi-domain dimeric cytoplasmic histidine kinase called LOV-HK, which is a key blue light-activated virulence factor in this microorganism. The structural basis of the light activation mechanism of this protein remains unclear. In this work, full-length LOV-HK was cloned, expressed and purified. The protein was activated by light and crystallized under a controlled illumination environment. The merge of 14 individual native data sets collected on a single crystal resulted in a complete X-ray diffraction data set to a resolution of 3.70 A with over 2 million reflections. Crystals belong to space group P212121, with unit-cell parameters a =95.96, b =105.30, c =164.49 A with a dimer in the asymmetric unit. Molecular replacement with Phaser using the individual domains as search models allowed for the reconstruction of almost the whole protein. Very recently, improved LOV-HK crystals led to a 3.25-A resolution dataset. Refinement and model building is underway. This crystal model will represent one of the very few examples of a multi-domain histidine kinase with known structure.