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Abstract The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis.

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. 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.

The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

ABSTRACTHere, we report the draft genome sequence of Methylobacterium sp. strain V23, a bacterium isolated from accretion ice of the subglacial Lake Vostok (3,592 meters below the surface). This genome makes possible the study of ancient and psychrophilic genes and proteins from a subglacial environment isolated from the surface for at least 15 million years.

Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis.

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.

Sterol carrier protein 2 (SCP2), a small intracellular domain present in all forms of life, binds with high affinity a broad spectrum of lipids. Due to its involvement in the metabolism of long-chain fatty acids and cholesterol uptake, it has been the focus of intense research in mammals and insects; much less characterized are SCP2 from other eukaryotic cells and microorganisms. We report here the X-ray structure of Yarrowia lipolytica SCP2 (YLSCP2) at 2.2 Å resolution in complex with palmitic acid. This is the first fungal SCP2 structure solved, and it consists of the canonical five-stranded β-sheet covered on the internal face by a layer of five α-helices. The overall fold is conserved among the SCP2 family, however, YLSCP2 is most similar to the SCP2 domain of human MFE-2, a bifunctional enzyme acting on peroxisomal β-oxidation. We have identified the common structural elements defining the shape and volume of the large binding cavity in all species characterized. Moreover, we found that the cavity of the SCP2 domains is distinctly formed by carbon atoms, containing neither organized water nor rigid polar interactions with the ligand. These features are in contrast with those of fatty acid binding proteins, whose internal cavities are more polar and contain bound water. The results will help to design experiments to unveil the SCP2 function in very different cellular contexts and metabolic conditions.

Several biological activities depend on iron–sulfur clusters ([Fe-S]). Even though they are well-known in several organisms their function and metabolic pathway were poorly understood in the majority of the organisms. We propose to use the amoeba Dictyostelium discoideum, as a biological model to study the biosynthesis of [Fe-S] at the molecular, cellular and organism levels. First, we have explored the D. discoideum genome looking for genes corresponding to the subunits that constitute the molecular machinery for Fe-S cluster assembly and, based on the structure of the mammalian supercomplex and amino acid conservation profiles, we inferred the full functionality of the amoeba machinery. After that, we expressed the recombinant mature form of D. discoideum frataxin protein (DdFXN), the kinetic activator of this pathway. We characterized the protein and its conformational stability. DdFXN is monomeric and compact. The analysis of the secondary structure content, calculated using the far-UV CD spectra, was compatible with the data expected for the FXN fold, and near-UV CD spectra were compatible with the data corresponding to a folded protein. In addition, Tryptophan fluorescence indicated that the emission occurs from an apolar environment. However, the conformation of DdFXN is significantly less stable than that of the human FXN, (4.0 vs. 9.0 kcal mol−1, respectively). Based on a sequence analysis and structural models of DdFXN, we investigated key residues involved in the interaction of DdFXN with the supercomplex and the effect of point mutations on the energetics of the DdFXN tertiary structure. More than 10 residues involved in Friedreich’s Ataxia are conserved between the human and DdFXN forms, and a good correlation between mutational effect on the energetics of both proteins were found, suggesting the existence of similar sequence/function/stability relationships. Finally, we integrated this information in an evolutionary context which highlights particular variation patterns between amoeba and humans that may reflect a functional importance of specific protein positions. Moreover, the complete pathway obtained forms a piece of evidence in favor of the hypothesis of a shared and highly conserved [Fe-S] assembly machinery between Human and D. discoideum.

The C4 carbon concentrating mechanism relies on specialized enzymes that have evolved unique expression patterns and biochemical properties distinct to their ancestral housekeeping forms. In maize and sorghum, the evolution of C4-NADP-malic enzyme (C4-NADP-ME) involved gene duplication and neofunctionalization, leading to the emergence of two plastidic isoforms: C4-NADP-ME and nonC4-NADP-ME, each with distinct kinetic and structural features. While C4-NADP-ME functions primarily as a tetramer, nonC4-NADP-ME exists in an equilibrium between dimeric and tetrameric forms, favoring the dimer in solution. This study shows which evolutionary changes in amino acid sequences influence the structure and function of these isoforms. By integrating X-ray crystallography, cryo-electron microscopy, computational molecular modeling and targeted biochemical analysis of mutant and truncated protein variants, we identify crucial roles for the N- and C-terminal regions and specific amino acid residues in governing isoform oligomerization. Our results reveal that the N-terminal region is essential for stabilizing the dimeric form of nonC4-NADP-ME, whereas specific adaptive substitutions and interactions with the C-terminal region enhance the stability of the tetrameric state characteristic of the C4-adapted isoform. We propose that differences in the N-terminal domain between the C4 and nonC4 isoforms reflect distinct selective pressures, which have driven their evolutionary divergence to fulfill specialized cellular functions.Competing Interest StatementThe authors have declared no competing interest.

The fijivirus Mal de Río Cuarto virus (MRCV) causes a devastating maize disease. Its non-structural protein P9-1, which shows ATPase and RNA binding activities, is the major component of the intracellular viroplasm where virus replication takes place. Herein, we established that the 24 C-terminal residues (C-arm) of P9-1 are required for the formation of viroplasm-like structures (VLS) in vivo and for the protein multimerization in vitro. Employing an integrative structural approach, we found that the C-arm is dispensable for P9-1 dimer assembly, but essential for the formation of doughnut-shaped pentamers and hexamers of dimers (decamers and dodecamers). Both assemblies, larger than those reported for other reoviruses, contain disordered loops oriented towards the inner pore of the structures, where RNA binding sites and conditional proteasome-mediated degradation signals (PEST) were predicted. In vitro assays demonstrated that ssRNA binding is favored towards P9-1 (do)decamers over the dimeric ΔC-arm version. In addition, although both P9-1 and P9-1ΔC-arm catalyzed the hydrolysis of ATP with similar activity values, an RNA-stimulated ATPase activity was only observed in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. Computational studies revealed a stronger preference of phosphate moieties to the decamer in the pore and the C-arm regions, suggesting that the allosteric communication between ATP and RNA binding sites is favored with this protein arrangement. Overall, our work reveals the structural versatility of a major viroplasm protein providing unprecedented insights into fijivirus viroplasm assembly and function and establishes the structural basis for the development of antiviral strategies against the Mal de Río Cuarto crop disease. Competing Interest Statement The authors have declared no competing interest.