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

doi:10.1016/j.bbrep.2018.09.005

Abstract

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 Å with over 2 million reflections. Crystals belong to space group P212121, with unit-cell parameters a = 95.96, b = 105.30, c = 164.49 Å 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-Å 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.

Highlights

Brucella abortus is a Gram-negative intracellular bacterium that affects cattle causing brucellosis, a worldwide disease that can be transmitted to humans
This light-dependent virulence enhancement is mediated by a cytoplasmic sensor histidine kinase called LOV-HK, which is part of a two-component signal transduction system involved in the modulation of the general stress response in Brucella [2]
This domain is followed by PAS (Per-ArntSim, 172–266), with unknown function, and HK (Histidine Kinase, residues 267–489), which autophosphorylates upon light absorption at a conserved histidine residue (His288) through a bound ATP molecule [4]

Summary

Introduction

Brucella abortus is a Gram-negative intracellular bacterium that affects cattle causing brucellosis, a worldwide disease that can be transmitted to humans. It has been shown that exposure of B. abortus to visible light results in a 10-fold higher level of bacterial replication in mouse macrophages than the corresponding dark control [1] This light-dependent virulence enhancement is mediated by a cytoplasmic sensor histidine kinase called LOV-HK, which is part of a two-component signal transduction system involved in the modulation of the general stress response in Brucella [2]. We present the crystallization and initial X-ray data analysis of the illuminated form of LOV-HK and describe its phasing by molecular replacement using the available domain fragments as search models This model will greatly complement the existing knowledge of this complex system and will provide essential information regarding the activation of sensor HKs in general

Macromolecule production

Crystallization

Data collection and processing

Results and discussion