Journal of Molecular Biology
Volume 385, Issue 5, 6 February 2009, Pages 1433-1444
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Design and Signaling Mechanism of Light-Regulated Histidine Kinases

https://doi.org/10.1016/j.jmb.2008.12.017Get rights and content

Abstract

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ∼ 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40°–60° rotational movement within an α-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.

Introduction

Perceiving external stimuli and reacting to them are essential for the survival of all living organisms in a changing environment. Proteins involved in signal transduction are constructed in a modular fashion from individual domains1 that fall largely into two groups, output or effector domains that possess biological activity such as catalytic or DNA binding activity, and input or sensor domains that are sensitive to signals such as absorption of light or binding of a chemical ligand. Interactions between sensor and effector domains may allosterically regulate the activity of the effector domain. The recombination of these domains throughout evolution may have been instrumental in the development of ever more sophisticated signaling networks in higher organisms. Moreover, the modularity of these systems allows the design of novel signaling proteins and entire networks that detect and integrate various stimuli. Recent work demonstrates that the activity of proteins such as actin-regulatory proteins2 and mitogen-activated protein kinases3 can be reprogrammed by coupling them with novel signal sensing domains.

Signaling proteins containing Per–Arnt–Sim (PAS) sensor domains occur in all kingdoms of life4 and regulate processes as diverse as phototropism in higher plants,5 voltage-dependent gating of ion channels in humans,6 and nitrogen fixation in rhizobia.7 The core of PAS domains adopts a common globular fold in which α-helices are packed on either side of a five-stranded antiparallel β-sheet. The core is often flanked by N- or C-terminal α-helical extensions that are either packed on the core or extend from it.8, 9 Certain PAS domains bind a cofactor; those that bind flavin nucleotides are referred to as light-oxygen-voltage (LOV) domains.10 Different PAS sensor domains detect different signals such as chemical ligands, light and redox potential. Their versatility is also manifest in the wide range of effector domains whose activity they regulate such as kinases, transcription factors and phosphodiesterases. The contrast between the uniformity of sensor domains and the diversity of effector domains to which they are covalently linked argues against structure-based signal transduction mechanisms that depend on specific tertiary contacts between sensor and effector domains. Rather, the mechanism might involve signal-dependent, order–disorder transitions of protein segments, specifically the N- and C-terminal helices,8, 11 or signal-dependent quaternary structure changes.12, 13

Given the natural diversity of effector domains, could PAS domains also be used to construct novel sensor proteins whose effector domains react to desired stimuli? Specifically, are different PAS sensor domains functionally interchangeable? To address these questions, we replaced the heme-binding PAS sensor domain of FixL from Bradyrhizobium japonicum (FixL), which confers oxygen sensitivity on its histidine kinase activity,14, 15 with the LOV blue light sensor domain of Bacillus subtilis YtvA (YtvA).16 Certain of the resulting fusion proteins retain kinase activity and substrate affinity in vitro fully comparable to that of FixL, but are regulated by blue light instead of by oxygen. Further, we demonstrate that these fusion proteins are also active in vivo and drive light-dependent gene expression in Escherichia coli. Kinase activity and its regulation by light depend critically on the nature of the linker between the PAS sensor and histidine kinase effector domains. We provide design rules to generate similar light-regulated kinases and other proteins.

Section snippets

Design of fusion proteins

B. subtilis YtvA comprises an N-terminal LOV sensor domain and a C-terminal STAS (sulfate transport antisigma factor antagonist) effector domain,17 joined by an α-helical linker sequence denoted Jα (Fig. 1a).16 In the dark, its LOV domain binds flavin mononucleotide (FMN) noncovalently. Absorption of blue light promotes formation of a covalent bond between the flavin ring and the conserved cysteine 62 within the LOV domain.18 This light-activated state thermally decays to the ground, dark state

The linker between the sensor and effector domains forms an α-helical coiled coil

In the structure of the isolated YtvA LOV domain,13 the C-terminal Jα linker forms an α-helix. Although the structure of the histidine kinase domain of FixL is not known, that of the homologous histidine kinase HK853 from Thermotoga maritima shows that the N-terminus of the DHp subdomain also forms an α-helix.30 We propose that the linker between the sensor and effector domains within our fusion proteins is formed by uniting these two shorter helices into a long, continuous, signaling α-helix.31

Molecular biology and protein purification

Genes encoding YtvA, FixL and FixJ were amplified via PCR and cloned into pET28c vectors. Fusion constructs were generated by fusion PCR, overlap-extension PCR and site-directed mutagenesis. Proteins were purified similar to the protocol described in Ref. 13. Concentrations were determined using extinction coefficients of 12,500 M 1 cm 1 at 450 nm for the fusion proteins, 1.26 × 105 M 1 cm 1 at 395 nm for FixL and 4860 M 1 cm 1 at 280 nm for FixJ.

Phosphorylation assays

Kinase activity assays were conducted by adapting

Acknowledgements

We thank Drs. Hauke Hennecke and Hans-Martin Fischer (ETH Zürich) and Dr. Michael Sadowsky (University of Minnesota) for supplying bacterial strains and plasmids. Drs. Tobin Sosnick, Sean Crosson and Michael Elowitz (CalTech) are acknowledged for providing materials, advice and comments on the manuscript. We gratefully appreciate the use of facilities of Drs. Francisco Bezanilla, Sean Crosson, Phoebe Rice and Tobin Sosnick. Laura Satkamp provided advice on fusion PCR, and Clark Hyde helped with

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