Jimena Rinaldi

Structure of the Full-Length Bacteriophytochrome from the Plant Pathogen Xanthomonas campestris Provides Clues to its Long-Range Signaling Mechanism

Phytochromes constitute a major superfamily of light-sensing proteins that are reversibly photoconverted between a red-absorbing (Pr) and a far-red-absorbing (Pfr) state. Bacteriophytochromes (BphPs) are found among photosynthetic and non-photosynthetic bacteria, including pathogens. To date, several BphPs have been biophysically characterized. However, it is still not fully understood how structural changes are propagated from the photosensory module to the output module during the signal transduction event. Most phytochromes share a common architecture consisting of an N-terminal photosensor that includes the PAS2-GAF-PHY domain triad and a C-terminal variable output module. Here we present the crystal structure of the full-length BphP from the plant pathogen Xanthomonas campestris pv. campestris (XccBphP) bearing its photosensor and its complete output module, a PAS9 domain. In the crystals, the protein was found to be in the Pr state, whereas diffraction data together with resonance Raman spectroscopic and theoretical results indicate a ZZZssa and a ZZEssa chromophore configuration corresponding to a mixture of Pr and Meta-R state, the precursor of Pfr. The XccBphP quaternary assembly reveals a head-to-head dimer in which the output module contributes to the helical dimer interface. The photosensor, which is shown to be a bathy-like BphP, is influenced in its dark reactions by the output module. Our structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module. This work contributes to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.

The β-scaffold of the LOV domain of the Brucella light-activated histidine kinase is a key element for signal transduction.

Light-oxygen-voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/β PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic β-scaffold in an antiparallel way. Our results point to the β-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.

A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyers patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

Crystallization and preliminary X‐ray characterization of the full‐length bacteriophytochrome from the plant pathogen Xanthomonas campestris pv. campestris

Phytochromes give rise to the largest photosensor family known to date. However, they are underrepresented in the Protein Data Bank. Plant, cyanobacterial, fungal and bacterial phytochromes share a canonical architecture consisting of an N-terminal photosensory module (PAS2-GAF-PHY domains) and a C-terminal variable output module. The bacterium Xanthomonas campestris pv. campestris, a worldwide agricultural pathogen, codes for a single bacteriophytochrome (XccBphP) that has this canonical architecture, bearing a C-terminal PAS9 domain as the output module. Full-length XccBphP was cloned, expressed and purified to homogeneity by nickel-NTA affinity and size-exclusion chromatography and was then crystallized at room temperature bound to its cofactor biliverdin. A complete native X-ray diffraction data set was collected to a maximum resolution of 3.25 Å. The crystals belonged to space group P43212, with unit-cell parameters a = b = 103.94, c = 344.57 Å and a dimer in the asymmetric unit. Refinement is underway after solving the structure by molecular replacement.

The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain.

The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.

Conformational plasticity of the intrinsically disordered protein ASR1 modulates its function as a drought stress-responsive gene

Plants in arid zones are constantly exposed to drought stress. The ASR protein family (Abscisic, Stress, Ripening) -a subgroup of the late embryogenesis abundant superfamily- is involved in the water stress response and adaptation to dry environments. Tomato ASR1, as well as other members of this family, is an intrinsically disordered protein (IDP) that functions as a transcription factor and a chaperone. Here we employed different biophysical techniques to perform a deep in vitro characterization of ASR1 as an IDP and showed how both environmental factors and in vivo targets modulate its folding. We report that ASR1 adopts different conformations such as α-helix or polyproline type II in response to environmental changes. Low temperatures and low pH promote the polyproline type II conformation (PII). While NaCl increases PII content and slightly destabilizes α-helix conformation, PEG and glycerol have an important stabilizing effect of α-helix conformation. The binding of Zn2+in the low micromolar range promotes α-helix folding, while extra Zn2+ results in homo-dimerization. The ASR1-DNA binding is sequence specific and dependent on Zn2+. ASR1 chaperone activity does not change upon the structure induction triggered by the addition of Zn2+. Furthermore, trehalose, which has no effect on the ASR1 structure by itself, showed a synergistic effect on the ASR1-driven heat shock protection towards the reporter enzyme citrate synthase (CS). These observations prompted the development of a FRET reporter to sense ASR1 folding in vivo. Its performance was confirmed in Escherichia coli under saline and osmotic stress conditions, representing a promising probe to be used in plant cells. Overall, this work supports the notion that ASR1 plasticity is a key feature that facilitates its response to drought stress and its interaction with specific targets.

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

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.

Study of a TCS activated by light in Brucella abortus

Two-component signal transduction systems (TCSs) are modules that allow bacteria to rapidly adapt to changing environmental conditions. In the most common case, they are formed by a sensor histidine kinase (HK) which, upon sensing of an external signal, autophosphorylates at a conserved histidine residue and then transfers the phosphoryl group to a conserved aspartate residue in a cognate response regulator (RR). The latter protein undergoes structural changes that are able to modify gene expression by directly binding to DNA, catalyze metabolic reactions or alter protein-protein interactions. The pathogenic bacterium Brucella abortus, the causative agent of the disease brucellosis, bears a particular two-component system formed by a dimeric cytoplasmic three-domain blue-light sensor HK (LOV-PAS-HK) and two monomeric RRs called PhyR and LovR. The activation of this HK has been shown to increase the virulence of this pathogen. With the goal of understanding at the atomic level the activation and signal transduction events of this system, we aimed to solve the threedimensional structures of these proteins by means of X-ray diffraction. The core of the blue-light sensor FMN-binding LOV domain was crystallized and its structure solved at 1.64 Å resolution in the dark. It adopts the alpha/beta PAS domain fold and presents a hydrophobic central beta-scaffold that interacts in one face with an FMN molecule and in the other with a neighboring monomer forming an unexpected antiparallel homodimer. This beta-scaffold destabilizes upon light exposure and therefore was proposed as a key element in the signal transduction mechanism [1]. Interestingly, we were also able to determine the structure of a construct comprising the LOV core domain plus an N-terminal capping helix at 2.34 Å resolution (N-LOV), observing now the expected parallel dimerization nature of the protein. This structure let us understand at the atomic level the important contribution of this N-terminal element in the stabilization of the quaternary structure and its photochemical behavior. This parallel arrangement has been recently confirmed with the determination of the N-LOV-PAS structure at 2.74 Å resolution, which holds a long connecting alpha-helical element between both globular domains. Additionally, we were able to solve the crystal structure of the isolated HK domain at 2.51 Å resolution by sulfur SAD in a challenging procedure, due to low sequence identity available models for MR, the low symmetry P2(1) space group present and the existence of four copies of the molecule in the 108-kDa asymmetric unit (AU) [2]. Interestingly, the HK structure presents two different dimeric assemblies in the AU, which allowed us to propose a mechanism of activation [3]. To finish, we were also able to determine the structure of the PhyR RR at 2.05 Å resolution. Efforts are underway to obtain the structure of the full LOV-PAS-HK protein as well as HK-RR complexes. All these protein structures, together with spectroscopic, activity and biophysical assays, allowed us a better understanding of this crucial system for the pathogenicity of Brucella. [1] Rinaldi, J. et al. (2012). J. Mol. Biol. 420, 112-127. [2] Klinke, S. et al. (2015). Acta Cryst. D71, 1433-1443. [3] Rinaldi, J. et al. (2016). J. Mol. Biol. 428, 1165-1179.