Dataset Information

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

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.

SUBMITTER: Rinaldi J

PROVIDER: S-EPMC6171537 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Rinaldi Jimena J Fernández Ignacio I Poth Lucía M LM Shepard William E WE Savko Martin M Goldbaum Fernando A FA Klinke Sebastián S

Biochemistry and biophysics reports 20180926

The pathogenic bacterium <i>Brucella abortus</i> 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 ...[more]

PMID: 30294679

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Project description:The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.

Dataset Information

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

Publications

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

Similar Datasets

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