Search

Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in Escherichia coli. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique dus gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using in vitro activity tests as well as in vivo E. coli complementation assays with the enzyme from Mycoplasma capricolum (DusB(MCap)), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusB(MCap) modifies U17 to D17 but also synthetizes D20/D20a combining therefore both E. coli DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the M. capricolum DusB enzyme could be an ancestral property.

International audience; Double stranded RNA-binding domain (dsRBD) is a ubiquitous domain specialized in the recognition of double-stranded RNAs (dsRNAs). Present in many proteins and enzymes involved in various functional roles of RNA metabolism, including RNA splicing, editing, and transport, dsRBD generally binds to RNAs that lack complex structures. However, this belief has recently been challenged by the discovery of a dsRBD serving as a major tRNA binding module for human dihydrouridine synthase 2 (hDus2), a flavoenzyme that catalyzes synthesis of dihydrouri-dine within the complex elbow structure of tRNA. We here unveil the molecular mechanism by which hDus2 dsRBD recognizes a tRNA ligand. By solving the crystal structure of this dsRBD in complex with a dsRNA together with extensive characterizations of its interaction with tRNA using mutagenesis, NMR and SAXS, we establish that while hDus2 dsRBD retains a conventional dsRNA recognition capability, the presence of an N-terminal extension appended to the canonical domain provides additional residues for binding tRNA in a structure-specific mode of action. Our results support that this extension represents a feature by which the dsRBD specializes in tRNA biology and more broadly highlight the importance of structural appendages to canonical domains in promoting the emergence of functional diversity.

Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π-π stacking interaction, involving a conserved tyrosine (Y346 in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C53). Here, the interaction between FAD and Y346 has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y346 mutants by ultrafast transient absorption spectroscopy. In C53A, the excited FAD very rapidly (0.43 ps) abstracts an electron from Y346, yielding the FAD˙-/Y346OH˙+ radical pair, while relaxation of the local environment (1.3 ps) of the excited flavin produces a slight Stokes shift of its stimulated emission band. The radical pair then decays via charge recombination, mostly in 3-4 ps, without any deprotonation of the Y346OH˙+ radical. Presumably, the H-bond between Y346 and the amide group of C53 increases the pKa of Y346OH˙+ and slows down its deprotonation. The dynamics of WT BsTrmFO shows additional slow decay components (43 and 700 ps), absent in the C53A mutant, assigned to excited FADox populations not undergoing fast photoreduction. Their presence is likely due to a more flexible structure of the WT protein, favored by the presence of C53. Interestingly, mutations of Y346 canceling its electron donating character lead to multiple slower quenching channels in the ps-ns regime. These channels are proposed to be due to electron abstraction either (i) from the adenine moiety of FAD, a distribution of the isoalloxazine-adenine distance in the absence of Y346 explaining the multiexponential decay, or (ii) from the W286 residue, possibly accounting for one of the decays. This work supports the idea that H-bond-assisted π-π stacking controls TrmFO's active site dynamics, required for competent orientation of the reactive centers during catalysis.

The C5-methylation of uracil to form 5-methyluracil (m5U) is a ubiquitous base modification of nucleic acids. Four enzyme families have converged to catalyze this methylation using different chemical solutions. Here, we investigate the evolution of 5-methyluracil synthase families in Mollicutes, a class of bacteria that has undergone extensive genome erosion. Many mollicutes have lost some of the m5U methyltransferases present in their common ancestor. Cases of duplication and subsequent shift of function are also described. For example, most members of the Spiroplasma subgroup, use the ancestral tetrahydrofolate-dependent TrmFO enzyme, to catalyze the formation of m5U54 in tRNA, while a TrmFO paralog (termed RlmFO) is responsible for m5U1939 formation in 23S RNA. RlmFO has replaced the S-adenosyl-l-methionine (SAM)-enzyme RlmD that adds the same modification in the ancestor and which is still present in mollicutes from the Hominis subgroup. Another paralog of this family, the TrmFO-like protein, has a yet unidentified function that differs from the TrmFO and RlmFO homologs. Despite having evolved towards minimal genomes, the mollicutes possess a repertoire of m5U modifying enzymes that is highly dynamic and has undergone horizontal transfer. This emphasizes the necessity for combining bioinformatics predictions with empirical testing and structural information to get a reliable functional annotation of these enzymes.

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK. Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli. Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both. Agence Nationale de la Recherche ANR-15-CE11-0001-02 Centre National de la Recherche Scientifique PICS07279 French State Program "Investissements d'Avenir" ANR-11-LABX-0011

International audience; Post-transcriptional base modifications are important to the maturation process of transfer RNAs (tRNAs). Certain modifications are abundant and present at several positions in tRNA as for example the dihydrouridine, a modified base found in the three domains of life. Even though the function of dihydrourine is not well understood, its high content in tRNAs from psychrophilic bacteria or cancer cells obviously emphasizes a central role in cell adaptation. The reduction of uridine to dihydrouridine is catalyzed by a large family of flavoenzymes named dihydrouridine synthases (Dus). Prokaryotes have three Dus (A, B and C) wherein DusB is considered as an ancestral protein from which the two others derived via gene duplications. Here, we unequivocally established the complete substrate specificities of the three Escherichia coli Dus and solved the crystal structure of DusB, enabling for the first time an exhaustive structural comparison between these bacterial flavoenzymes. Based on our results, we propose an evolutionary scenario explaining how substrate specificities has been diversified from a single structural fold.

International audience; To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.

Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.

International audience; In most organisms, the widely conserved 1-methyl-adenosine58 (m(1)A58) tRNA modification is catalyzed by an S-adenosyl-L-methionine (SAM)-dependent, site-specific enzyme TrmI. In archaea, TrmI also methylates the adjacent adenine 57, m(1)A57 being an obligatory intermediate of 1-methyl-inosine57 formation. To study this multi-site specificity, we used three oligoribonucleotide substrates of Pyrococcus abyssi TrmI (PabTrmI) containing a fluorescent 2-aminopurine (2-AP) at the two target positions and followed the RNA binding kinetics and methylation reactions by stopped-flow and mass spectrometry. PabTrmI did not modify 2-AP but methylated the adjacent target adenine. 2-AP seriously impaired the methylation of A57 but not A58, confirming that PabTrmI methylates efficiently the first adenine of the A57A58A59 sequence. PabTrmI binding provoked a rapid increase of fluorescence, attributed to base unstacking in the environment of 2-AP. Then, a slow decrease was observed only with 2-AP at position 57 and SAM, suggesting that m(1)A58 formation triggers RNA release. A model of the protein-tRNA complex shows both target adenines in proximity of SAM and emphasizes no major tRNA conformational change except base flipping during the reaction. The solvent accessibility of the SAM pocket is not affected by the tRNA, thereby enabling S-adenosyl-L-homocysteine to be replaced by SAM without prior release of monomethylated tRNA.

Enzyme-catalyzed reactions often rely on a noncovalently bound cofactor whose reactivity is tuned by its immediate environment. Flavin cofactors, the most versatile catalyst encountered in biology, are often maintained within the protein throughout numbers of complex ionic and aromatic interactions. Here, we have investigated the role of π-π stacking and hydrogen bond interactions between a tyrosine and the isoalloxazine moiety of the flavin adenine dinucleotide (FAD) in an FAD-dependent RNA methyltransferase. Combining several static and time-resolved spectroscopies as well as biochemical approaches, we showed that aromatic stacking is assisted by a hydrogen bond between the phenol group and the amide of an adjacent active site loop. A mechanism of recognition and binding of the redox cofactor is proposed.

The techniques of high applied pressure and flash photolysis have been combined to study ligand rebinding to neuroglobin (Ngb) and tomato Hb, globins that may display a His-Fe-His hexacoordination in the absence of external ligands. High pressure induces a moderate decrease in the His association rate and a large decrease in His dissociation rate, thus leading to an enhancement of the overall His affinity. The overall structural difference between penta- and hexacoordinated globins may be rather small and can be overcome by external modifications such as high pressure. Over the pressure range 0.1-700 MPa (7 kbar), the globins may show a loss of over a factor of 100 in the amplitude of the bimolecular rebinding phase after photodissociation. The kinetic data show that pressure induces a moderate increase of the rate for ligand binding from the correlated pair state (just after photodissociation) and a large (factor of 1000) decrease in rate for migration through the protein. The effect on the ligand migration phase was similar for both the external ligands (such as oxygen) as for the internal (histidine) ligand, suggesting the dominant role of protein fluctuations, rather than specific chemical barriers. Thus high pressure efficiently closes the protein migration channels; however, contrary to the effect of high viscosity, high pressure induces a greater decrease in rate for ligand migration toward the exterior (heme to the solvent) versus inward migration, as if the presence of the ligand itself induces an additional steric constraint.

International audience; Neuroglobin plays an important function in the supply of oxygen in nervous tissues. In human neuroglobin, a cysteine at position 46 in the loop connecting the C and D helices of the globin fold is presumed to form an intramolecular disulfide bond with Cys55. Rupture of this disulfide bridge stabilizes bi-­histidyl haem hexacoordination, causing an overall decrease in the affinity for oxygen. Here, the first X-ray structure of wild-type human neuroglobin is reported at 1.74 Å resolution. This structure provides a direct observation of two distinct conformations of the CD region containing the intramolecular disulfide link and highlights internal cavities that could be involved in ligand migration and/or are necessary to enable the conformational transition between the low and high oxygen-affinity states following S—S bond formation.

International audience; Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the T-Psi-C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein. Cop. 2010 Elsevier Inc. All rights reserved.

International audience; Neuroglobin (Ngb) and the cellular prion protein (PrPc), proteins of unknown function in the nervous system, are known to be expressed in the retina and have been observed in different rat retinal cells. The retina is the site of the highest concentration for Ngb, a heme protein of similar size and conformation to myoglobin. In this study, we demonstrated by immunohistochemical analysis of retinal colocalization of Ngb and PrPc in the ganglion cell layer. Considering for these two a common protective role in relation to oxidative stress and a possible transient contact during migration of PrPc through the eye or upon neuronal degradation, we undertook in vitro studies of the interaction of the purified proteins. Mixing these two proteins leads to rapid aggregation, even at submicromolar concentrations. As observed with the use of dynamic light scattering, particles comprising both proteins evolve to hundreds of nanometers within several seconds, a first report showing that PrPc is able to form aggregates without major structural changes. The main effect would then appear to be a protein–protein interaction specific to the surface charge of the Ngb protein with PrPc Nterminal sequence. A dominant parameter is the solvent ionic force, which can significantly modify the final state of aggregation. PrPc, normally anchored to the cell membrane, is toxic in the cytoplasm, where Ngb is present; this could suggest an Ngb function of scavenging proteins capable of forming deleterious aggregates considering a charge complementarity in the complex.

International audience; The purification, crystallization and successful structure determination by molecular replacement of wild-type human brain neuroglobin at 1.8 Å resolution is reported. The apparent space group was orthorhombic C2221, but the real space group was monoclinic P21, which resulted from twinning. Indeed, the unit-cell parameters, a = 31.2, b = 139.1, c = 31.2 Å, β = 102°, display a fortuitously close to c and twinning by the operator l, −k, h occurs. Twinning was not evident from the initial analysis of intensity distribution, but pseudo-merohedral twinning was revealed by the Padilla and Yeates test based on local intensity differences. A twinning fraction of 0.5 was determined in SHELXL, indicating a perfect hemihedrally twinned crystal. To date, this type of twinning has been reported in more than ten structures, which makes it quite a common case in proteins.

We describe a software program to help exploit a database of aligned protein sequences. In addition to the classical lists of sequences, a graphical representation is used to get a better overview of the information. As natural parameters, the type of amino acid and sequence position are used. Various plots or 3D representations are then updated. Examples are shown based on globin sequences from various species and on the abnormal human hemoglobins. The software should be of interest to protein engineers who need to know what variants are already known.

Neuroglobin (Ngb) and cytoglobin (Cygb), recent additions to the globin family, display a hexa-coordinated (bis-histidyl) heme in the absence of external ligands. Although these proteins have the classical globin fold they reveal a very high thermal stability with a melting temperature (Tm) of 100 degrees C for Ngb and 95 degrees C for Cygb. Moreover, flash photolysis experiments at high temperatures reveal that Ngb remains functional at 90 degrees C. Human Ngb may have a disulfide bond in the CD loop region; reduction of the disulfide bond increases the affinity of the iron atom for the distal (E7) histidine, and leads to a 3 degrees C increase in the T(m) for ferrous Ngb. A similar Tm is found for a mutant of human Ngb without cysteines. Apparently, the disulfide bond is not involved directly in protein stability, but may influence the stability indirectly because it modifies the affinity of the distal histidine. Mutation of the distal histidine leads to lower thermal stability, similar to that for other globins. Only globins with a high affinity of the distal histidine show the very high thermal stability, indicating that stable hexa-coordination is necessary for the enhanced thermal stability; the CD loop which contains the cysteines appears as a critical region in the neuroglobin thermal stability, because it may influence the affinity of the distal histidine.

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.