Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes

The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity

The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis

Insights into the hyperthermostability and unusual region-specificity of archaeal Pyrococcus abyssi tRNA m1A57/58 methyltransferase

The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence

Prediction of the Iron–Sulfur Binding Sites in Proteins Using the Highly Accurate Three-Dimensional Models Calculated by AlphaFold and RoseTTAFold

AlphaFold and RoseTTAFold are deep learning-based approaches that predict the structure of proteins from their amino acid sequences. Remarkable success has recently been achieved in the prediction accuracy of not only the fold of the target protein but also the position of its amino acid side chains. In this article, I question the accuracy of these methods to predict iron–sulfur binding sites. I analyze three-dimensional models calculated by AlphaFold and RoseTTAFold of Fe–S–dependent enzymes, for which no structure of a homologous protein has been solved experimentally. In all cases, the amino acids that presumably coordinate the cluster were gathered together and facing each other, which led to a quite accurate model of the Fe–S cluster binding site. Yet, cysteine candidates were often involved in intramolecular disulfide bonds, and the number and identity of the protein amino acids that should ligate the cluster were not always clear. The experimental structure determination of the protein with its Fe–S cluster and in complex with substrate/inhibitor/product is still needed to unambiguously visualize the coordination state of the cluster and understand the conformational changes occurring during catalysis

Etude biochimique et structurale de deux pyrimidine-c5 méthyltransférases des arn de transfert

Dans la cellule, l ARNt est une molécule-clé de la traduction génétique. Pour être fonctionnel, il doit subir différentes étapes de maturation post-transcriptionnelle, au cours desquelles certains de ses nucléotides sont modifiés chimiquement grâce à des enzymes dites de modification. Mon sujet de thèse a porté sur l étude biochimique et structurale de deux pyrimidine-C5 méthyltransférases (MTases) des ARNt. Une première partie de mon travail a consisté en la caractérisation biochimique de la cytosine-C5 MTase de S. cerevisiae, Trm4. L analyse de son mécanisme catalytique et de son organisation modulaire a ainsi été réalisée. Une seconde partie de mon travail a contribué à l identification de la m5U54 MTase d ARNt de P. abyssi, PabTrmU54, et a conduit à la résolution de sa structure tridimensionnelle en complexe avec la S-adénosyl-L-homocystéine, par cristallographie aux rayons X. L ensemble de ces résultats a permis d améliorer nos connaissances sur le mode de reconnaissance spécifique du substrat ARN par les enzymes de modificationIn the cell, tRNA is a key molecule of genetic translation. To become functional, it undergoes different steps of post-transcriptional maturation. During this process, some of its nucleosides are chemically modified by modification enzymes. My thesis project focused on the biochemical and structural study of two tRNA C5-pyrimidine methyltransferases (MTases). The first part of my work consisted in the biochemical characterization of the S. cerevisiae C5-cytosine MTase, Trm4. The analysis of its catalytic mechanism and of its modular organization was then realized. The second part of my work contributed to the identification of the P. abyssi tRNA m5U54 MTase, PabTrmU54, and led to the resolution of its crystal structure in complex with S-adenosyl-L-homocysteine, by X-ray crystallography. Finally, all these results participated in the improvement of our knowledge about the specific mode of RNA recognition by modification enzymesORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Ordering of C-terminal Loop and Glutaminase Domains of Glucosamine-6-Phosphate Synthase Promotes Sugar Ring Opening and Formation of the Ammonia Channel

International audienc

Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase

Sequence comparison of several RNA m5C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m5C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m5C967 methyltransferase, but whether this conclusion applies to other RNA m5C methyltransferases remains to be verified. Yeast tRNA m5C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex

A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis

International audienceThe flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH2THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH• radical, and a catalytic intermediate endowed with methylating activity. The FADH• radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH2THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH2THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH2THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions

Structural comparison of tRNA m¹A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions

<p>Abstract</p> <p>Background</p> <p>tRNA m¹A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m¹A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.</p> <p>Results</p> <p>In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal <it>P. abyssi </it>enzyme is strengthened by four intersubunit disulfide bridges.</p> <p>Conclusions</p> <p>The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.</p

Pseudo-merohedral twinning in monoclinic crystals of wild-type human brain neuroglobin

International audienceThe 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