Dynamic interplays between three redox cofactors in a DNA photolyase revealed by spectral decomposition.

DNA repair catalyzed by photolyases is accomplished by a light-dependent electron transfer event from a fully reduced flavin adenine dinucleotide to a DNA lesion site. Prokaryotic DNA photolyase, PhrB, possesses a ribolumazine cofactor and a four-iron-four-sulfur cluster in addition to the catalytic flavin, but their functional roles are poorly understood. Here, we employ time-resolved absorption spectroscopy to probe light-induced responses in both solution and single crystals of PhrB. We jointly analyze a large collection of light-induced difference spectra from the wild-type and mutant PhrB obtained under different light and redox conditions. By applying singular value decomposition to 159 time series, we dissect light-induced spectral changes and examine the dynamic interplay between three cofactors. Our findings suggest that these cofactors form an interdependent redox network to coordinate light-induced redox responses. We propose that the ribolumazine cofactor serves as a photoprotective pigment under intense light or prolonged illumination, while the iron-sulfur cluster acts as a transient electron cache to maintain balance between two otherwise independent photoreactions of the flavin and ribolumazine.

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 including DNA processing. The prokaryotic DNA repair enzyme PhrB, a member of the protein family of cryptochromes and photolyases, carries a four-iron-four-sulfur cluster [4Fe4S] in addition to the catalytic cofactor flavin adenine dinucleotide (FAD) and a second pigment 6,7-dimethyl-8-ribityllumazine (DMRL). The light-induced redox reactions of this multi-cofactor protein complex were recently shown as two interdependent photoreductions of FAD and DMRL mediated by the [4Fe4S] cluster functioning as an electron cache to hold a fine balance of electrons. Here, we apply the more traditional temperature-scan cryo-trapping technique in protein crystallography and the newly developed technology of in situ serial Laue diffraction at room temperature. These diffraction methods in dynamic crystallography enable us to capture strong signals of electron density changes in the [4Fe4S] cluster that depict quantized electronic movements. The mixed valence layers of the [4Fe4S] cluster due to spin coupling and their dynamic responses to light illumination are observed directly in our difference maps between its redox states. These direct observations of the quantum effects in a protein bound iron-sulfur cluster have thus opened a window into the mechanistic understanding of metal clusters in biological systems.

Dimer Asymmetry and Light Activation Mechanism in Brucella Blue-Light Sensor Histidine Kinase.

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.

Crystal-on-crystal chips for in situ serial diffraction at room temperature.

Recent developments in serial crystallography at X-ray free electron lasers (XFELs) and synchrotrons have been driven by two scientific goals in structural biology - first, static structure determination from nano or microcrystals of membrane proteins and large complexes that are difficult for conventional cryocrystallography, and second, direct observations of transient structural species in biochemical reactions at near atomic resolution. Since room-temperature diffraction experiments naturally demand a large quantity of purified protein, sample economy is critically important for all steps of serial crystallography from crystallization, crystal delivery to data collection. Here we report the development and applications of "crystal-on-crystal" devices to facilitate large-scale in situ serial diffraction experiments on protein crystals of all sizes - large, small, or microscopic. We show that the monocrystalline quartz as a substrate material prevents vapor loss during crystallization and significantly reduces background X-ray scattering. These devices can be readily adopted at XFEL and synchrotron beamlines, which enable efficient delivery of hundreds to millions of crystals to the X-ray beam, with an overall protein consumption per dataset comparable to that of cryocrystallography.