Structural basis of molecular logic OR in a dual-sensor histidine kinase.

Signal detection and integration by sensory proteins constitute the critical molecular events as living organisms respond to changes in a complex environment. Many sensory proteins adopt a modular architecture that integrates the perception of distinct chemical or physical signals and the generation of a biological response in the same protein molecule. Currently, how signal perception and integration are achieved in such a modular, often dimeric, framework remains elusive. Here, we report a dynamic crystallography study on the tandem sensor domains of a dual-sensor histidine kinase PPHK (phosphorylation-responsive photosensitive histidine kinase) that operates a molecular logic OR, by which the output kinase activity is modulated by a phosphorylation signal and a light signal. A joint analysis of ∼170 crystallographic datasets probing different signaling states shows remarkable dimer asymmetry as PPHK responds to the input signals and transitions from one state to the other. Supported by mutational data and structural analysis, these direct observations reveal the working mechanics of the molecular logic OR in PPHK, where the light-induced bending of a long signaling helix at the dimer interface is counteracted by the ligand-induced structural changes from a different sensor domain. We propose that the logic OR of PPHK, together with an upstream photoreceptor, implements a "long-pass" red light response distinct from those accomplished by classical phytochromes.

Photoactivation mechanism of a carotenoid-based photoreceptor.

Photoprotection is essential for efficient photosynthesis. Cyanobacteria have evolved a unique photoprotective mechanism mediated by a water-soluble carotenoid-based photoreceptor known as orange carotenoid protein (OCP). OCP undergoes large conformational changes in response to intense blue light, and the photoactivated OCP facilitates dissipation of excess energy via direct interaction with allophycocyanins at the phycobilisome core. However, the structural events leading up to the OCP photoactivation remain elusive at the molecular level. Here we present direct observations of light-induced structural changes in OCP captured by dynamic crystallography. Difference electron densities between the dark and illuminated states reveal widespread and concerted atomic motions that lead to altered protein-pigment interactions, displacement of secondary structures, and domain separation. Based on these crystallographic observations together with site-directed mutagenesis, we propose a molecular mechanism for OCP light perception, in which the photochemical property of a conjugated carbonyl group is exploited. We hypothesize that the OCP photoactivation starts with keto-enol tautomerization of the essential 4-keto group in the carotenoid, which disrupts the strong hydrogen bonds between the bent chromophore and the protein moiety. Subsequent structural changes trapped in the crystal lattice offer a high-resolution glimpse of the initial molecular events as OCP begins to transition from the orange-absorbing state to the active red-absorbing state.

Spin-Coupled Electron Densities of Iron-Sulfur Cluster Imaged by In Situ Serial Laue Diffraction.

Iron-sulfur clusters are inorganic cofactors found in many proteins involved in fundamental biological processes. The prokaryotic DNA repair photolyase PhrB carries a four-iron-four-sulfur cluster ([4Fe4S]) in addition to the catalytic flavin adenine dinucleotide (FAD) and a second cofactor ribolumazine. Our recent study suggested that the [4Fe4S] cluster functions as an electron cache to coordinate two interdependent photoreactions of the FAD and ribolumazine. Here we report the crystallography observations of light-induced responses in PhrB using the cryo-trapping method and in situ serial Laue diffraction at room temperature. We capture strong signals that depict electron density changes arising from quantized electronic movements in the [4Fe4S] cluster. Our data reveal the mixed valence layers of the [4Fe4S] cluster due to spin coupling and their dynamic responses to light-induced redox changes. The quantum effects imaged by decomposition of electron density changes have shed light on the emerging roles of metal clusters in proteins.

Resolution of structural heterogeneity in dynamic crystallography.

Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods.

Dynamic Crystallography Reveals Early Signalling Events in Ultraviolet Photoreceptor UVR8.

Arabidopsis thaliana UVR8 (AtUVR8) is a long-sought-after photoreceptor that undergoes dimer dissociation in response to UV-B light. Crystallographic and mutational studies have identified two crucial tryptophan residues for UV-B responses in AtUVR8. However, the mechanism of UV-B perception and structural events leading up to dimer dissociation remain elusive at the molecular level. We applied dynamic crystallography to capture light-induced structural events in photoactive AtUVR8 crystals. Here we report two intermediate structures at 1.67Å resolution. At the epicenter of UV-B signaling, concerted motions associated with Trp285/Trp233 lead to ejection of a water molecule, which weakens an intricate network of hydrogen bonds and salt bridges at the dimer interface. Partial opening of the ß-propeller structure due to thermal relaxation of conformational strains originating in the epicenter further disrupts the dimer interface and leads to dimer dissociation. These dynamic crystallographic observations provide structural insights into the photo-perception and signaling mechanism of UVR8.