Lisandro H. Otero

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.

Snapshots of Conformational Changes Shed Light Into the Ntrx Receiver Domain Signal Transduction Mechanism

Brucella abortus is an important pathogenic bacterium that has to overcome oxygen deficiency in order to achieve a successful infection. Previously, we proved that a two-component system formed by the histidine kinase NtrY and the response regulator NtrX is essential to achieve an adaptive response to low oxygen tension conditions. Even though the relevance of this signaling pathway has already been demonstrated in other microorganisms, its molecular activation mechanism has not yet been described in detail. In this article, we report the first crystal structures from different conformations of the NtrX receiver domain from B. abortus, and we propose a sequence of events to explain the structural rearrangements along the activation process. The analysis of the structures obtained in the presence of the phosphoryl group analog beryllofluoride led us to postulate that changes in the interface formed by the α4 helix and the β5 strand are important for the activation, producing a reorientation of the α5 helix. Also, a biochemical characterization of the NtrX receiver domain enzymatic activities was performed, describing its autophosphorylation and autodephosphorylation kinetics. Finally, the role of H85, an important residue, was addressed by site-directed mutagenesis. Overall, these results provide significant structural basis for understanding the response regulator activation in this bacterial two-component system.

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand-alone TPM domain.

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X‐ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand‐alone TPM domain. The structure reveals a new topological variant of the four β‐strands constituting the central β‐sheet of the αβα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42. Proteins 2014; 82:3062–3078.

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 disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N‐acetylglucosamine (NAG) and N‐acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell‐wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three‐dimensional structure of a lysozyme in complex with bacterial cell‐wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival.

Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins.

Structural and functional characterization of a cold adapted TPM-domain with ATPase/ADPase activity.

The Pfam PF04536 TPM_phosphatase family is a broadly conserved family of domains found across prokaryotes, plants and invertebrates. Despite having a similar protein fold, members of this family have been implicated in diverse cellular processes and found in varied subcellular localizations. Very recently, the biochemical characterization of two evolutionary divergent TPM domains has shown that they are able to hydrolyze phosphate groups from different substrates. However, there are still incorrect functional annotations and uncertain relationships between the structure and function of this family of domains. BA41 is an uncharacterized single-pass transmembrane protein from the Antarctic psychrotolerant bacterium Bizionia argentinensis with a predicted compact extracytoplasmic TPM domain and a C-terminal cytoplasmic low complexity region. To shed light on the structural properties that enable TPM domains to adopt divergent roles, we here accomplish a comprehensive structural and functional characterization of the central TPM domain of BA41 (BA41-TPM). Contrary to its predicted function as a beta-propeller methanol dehydrogenase, light scattering and crystallographic studies showed that BA41-TPM behaves as a globular monomeric protein and adopts a conserved Rossmann fold, typically observed in other TPM domain structures. Although the crystal structure reveals the conservation of residues involved in substrate binding, no putative catalytic or intramolecular metal ions were detected. Most important, however, extensive biochemical studies demonstrated that BA41-TPM has hydrolase activity against ADP, ATP, and other di- and triphosphate nucleotides and shares properties of cold-adapted enzymes. The role of BA41 in extracellular ATP-mediated signaling pathways and its occurrence in environmental and pathogenic microorganisms is discussed.

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.