Dataset Information

Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

ABSTRACT: Histidine protein kinases (HKs) are prevalent prokaryotic sensor kinases that are central to phosphotransfer in two-component signal transduction systems, regulating phosphorylation of response regulator proteins that determine the output responses. HKs typically exist as dimers and can potentially autophosphorylate at each conserved histidine residue in the individual protomers, leading to diphosphorylation. However, analyses of HK phosphorylation in biochemical assays in vitro suggest negative cooperativity, whereby phosphorylation in one protomer of the dimer inhibits phosphorylation in the second protomer, leading to ?50% phosphorylation of the available sites in dimers. This negative cooperativity is often correlated with an asymmetric domain arrangement, a common structural characteristic of autophosphorylation states in many HK structures. In this study, we engineered covalent dimers of the cytoplasmic domains of Escherichia coli CpxA, enabling us to quantify individual species: unphosphorylated, monophosphorylated, and diphosphorylated dimers. Together with mathematical modeling, we unambiguously demonstrate no cooperativity in autophosphorylation of CpxA despite its asymmetric structures, indicating that these asymmetric domain arrangements are not linked to negative cooperativity and hemiphosphorylation. Furthermore, the modeling indicated that many parameters, most notably minor amounts of ADP generated during autophosphorylation reactions or present in ATP preparations, can produce ?50% total phosphorylation that may be mistakenly attributed to negative cooperativity. This study also establishes that the engineered covalent heterodimer provides a robust experimental system for investigating cooperativity in HK autophosphorylation and offers a useful tool for testing how symmetric or asymmetric structural features influence HK functions.

SUBMITTER: Bouillet S

PROVIDER: S-EPMC7278341 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

Bouillet Sophie S Wu Ti T Chen Shaoxing S Stock Ann M AM Gao Rong R

The Journal of biological chemistry 20200224 23

Histidine protein kinases (HKs) are prevalent prokaryotic sensor kinases that are central to phosphotransfer in two-component signal transduction systems, regulating phosphorylation of response regulator proteins that determine the output responses. HKs typically exist as dimers and can potentially autophosphorylate at each conserved histidine residue in the individual protomers, leading to diphosphorylation. However, analyses of HK phosphorylation in biochemical assays <i>in vitro</i> suggest n ...[more]

PMID: 32094228

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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

Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

Publications

Structural asymmetry does not indicate hemiphosphorylation in the bacterial histidine kinase CpxA.

Similar Datasets

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