DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Biochemical and Structural characterization of MnmE/MnmG and GTPBP3/MTO1 protein complexes involved in redox modifications of bacterial and human tRNAs – MiGRATe

Biochemical and structural characterization of the enzymatic complexes MnmEG and GTPBP3/MTO1 involved in the redox modification of tRNAs in bacteria and in human

The aim of the project is to study the structure / function relationship of enzymatic complexes conserved throughout evolution (MnmE / MnmG in bacteria and GTPBP3 / MTO1 in humans) and involved in the 5-carboxyaminomethylation modification of tRNA wobble position that is necessary for the correct decoding of the genetic information. Indeed, the lack of modification leads to growth disorders in bacteria and severe pathologies in humans.

Understanding the molecular basis for the functioning of an enzymatic RNA modification complex

We choose to focus our project on MnmEG and GTPBP3/MTO1complexes for several reasons:<br />The reaction mechanism catalyzed by MnmEG is still a matter of discussion. Regarding GTPBP3/MTO1, nothing has been done in the biochemical and structural characterizations.<br />This reaction is performed by a protein complex that orchestrates a complicated catalysis by bringing about GDP, glycine (or taurine for the human complex), folate, flavin, NADH and the tRNA substrate. This exceptional complex reaction must be highly synchronized and we hypothesize that MnmE or GTPBP3, acting as a molecular switch, may control the generation of highly labile intermediate and their tunneling from one protein to another. Therefore, we anticipate new molecular mechanisms controlling the coupling between the chemical reaction notably throughout the G protein cycle of the GTPase activated by dimerization (GAD) belonging MnmE family and the protein/RNA conformational changes during catalysis.<br />We and others have established a physiological link between the activity of MnmEG and the inhibition of FeS-dependent MiaB, which is another evolutionary conserved tRNA modification enzyme that catalyzes the formation of ms2i6A37. Interestingly, YgfZ described as a folate binding protein shares with MnmE the same folate binding domain and this protein seems to protect the iron sulfur cluster of MiaB and others from deleterious uncoupled activity of MnmE. Therefore the interplay between the three proteins i.e YgfZ, MiaB and MnmEG as well as the role of folate in FeS assembly and repair deserved to be further investigated. <br />Finally, understanding the mechanism of cmnm5U formation and the regulatory process triggered by MnmEG in a model organism such as E. coli and that of tm5U in human will not only shed light on the mitochondrial diseases but also may enable to promote the discovery of future antibiotics that specifically target the MnmE/MnmG without affecting GTPBP3/MTO1.

To characterize the enzymes and interrogate their modes of action, we will use traditional biochemical and enzymological approaches in combination with structural methods such as X-ray crystallography and various forms of spectroscopy, as well as small-scale organic synthesis and fast-reaction kinetic methods. In addition we will investigate in vitro and in vivo notably by genetic methods, the new role of MnmEG in the iron sulfur cluster assembly of several enzymes. We will investigate the flavin and folate-dependent reaction mechanism of MnmEG from E. coli, which is still a matter of controversy. Our main purpose will be to understand: 1- What are the reactions catalyzed by the individual proteins MnmE and MnmG? 2-Why they both work in concert to modify the tRNA? Our recent knowledge on a homologous enzymatic system will considerably help us to elucidate the flavin reaction mechanism of MnmG. Because the reaction intermediates can be quite elusive in mesophilic organisms, the use of MnmE/G from thermophilic organisms could be a tremendous advantage to observe and trap labile intermediates. An important task will concern the structural aspects of the enzymatic system notably the determination of the crystal structure of the MnmEG complex. The main goal will be to unravel how MnmE and MnmG work together for catalyzing the modification of tRNA and try to uncover the conformational changes that lead to tunneling of intermediate. If we succeed in the crystallization, any structure could be solved using by molecular replacement using the individual structures of MnmE and MnmG. We also intend to understand the interplay between the three proteins YgfZ, MnmE, and MiaB from E. coli as well as the effect of folate on iron sulfur cluster assembly. Finally the expression and purification as well structural characterization of the GTPBP3/MTO1 will be investigated.

1-Mechanism of MnmEG: One of the hypotheses that we had formulated is that MnmEG could have a reaction intermediate common to that of the flavin-dependent tRNA methyltransferase TrmFO. This intermediate is proposed to be a flavin reduced with a methylene on the N5 atom of the redox nucleus of flavin, and would act as the nucleotide methylating agent. It has been proposed that the latter be transiently produced by the enzyme by consuming an NADH molecule to reduce the FAD and a molecule of methylenetetrahydrofolate (CH2THF) at the origin of CH2 grafted onto the C5 of uridine 34 and which would be the first step in the mechanism of modification of tRNA by the enzymatic complex, MnmEG and by TrmFO. We have for the first time succeeded in synthesizing this reaction intermediate from free flavin and a cheap chemical reagent and showed that this stable intermediate can activate an apoprotein form of TrmFO to methylate the tRNA, thereby bypassing the natural enzymatic pathway. We have also shown that this intermediate can bind to MnmG and to MnmEG complex.
2- Crystallization of the MnmE / MnmG complex of e. Coli: We were able to reconstitute a stable MnmEG complex by means of a coexpression of the two genes. This complex has been purified and crystallized. We do not know at this stage whether it is a crystal of the complex and not one of the proteins of the complex.
3- Studies of human proteins GTPBP3 / MTO1: We cloned the most abundant isoform of MTO1 and GTPBP3 and we tried to express them. Unfortunately, we did not have an expression of MTO1. On the other hand, we managed to express in large quantities GTPBP3 for our biochemical, biophysical and structural studies. We also succeeded in obtaining crystals of this protein.

The synthesis of a stable intermediate of the catalytic cycle is unique in the field of enzymology. In the near future we will try to demonstrate whether the latter, which activated TrmFO, can also activate the complex MnmEG and GTPBP3/MTO1. Another crucial point will be the continuation of the first encouraging results concerning the crystallization approaches of the bacterial MnmEG complex. The final step would be to obtain the structure of the complex. We will also focus on the structural resolution of human GTPBP3 as well as on the structural and biochemical study of the physiopathological mutations of the latter seen in patients suffering from cardiomyopathy and lactic acidosis. A major effort will be devoted to the development of human MTO1 expression strategy. In particular, the switch to yeast expression systems will also be considered. Other project objectives in addition to studying the effect of MnmEG on the assembly of MiaB sulfur iron cofactors will be investigated later.

A paper on the synthesis of the flavinic catalytic intermediate with a nucleoside methylation capacity and the activation of the MnmG homologue by the latter for methylation of the tRNA has been submitted to NATURE journal.

Post-transcriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information encoded by DNA. The chemical modifications of RNA span all three phylogenetic domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. Among the RNA species, tRNAs are the most heavily modified. Approximately 15%–25% of all nucleosides in eukaryotic tRNA contain modifications. Although not fully understood, these modifications have been proposed to serve various purposes: (1) tRNA discrimination (e.g., initiator tRNAMet is distinguished from elongator tRNAMet through ribosylation at A64); (2) translation fidelity, where absence of modifications at wobble position 34 causes decoding errors because the modified base either restrict or extends codon-anticodon interaction capability through base pairing; and (3) tRNA stability and quality control of the macromolecule. To date more than a hundred different types of modifications have been reported and more will certainly be discovered in the future, thanks to new RNA sequencing technologies. The chemical diversity of these “new nucleotides” is astonishing and puzzling since no other biological macromolecule is subjected to such enormous biochemical transformation. Recently spectacular mechanisms and unprecedented chemistry have been unraveled. Besides the chemistry, the finding that catalysis of complicated RNA modifications can be orchestrated by complex edifices with the participation of several proteins render the world of RNA enzymology more appealing and challenging for biophysical and structural characterizations. This is the case of MnmEG hetero-complex that catalyzes the complex formation of the evolutionary conserved 5-carboxymethylaminouridine (cmnm5U or cmnm5s2U) at the wobble uridine of several Escherichia coli tRNAs. In mitochondria, owing to their bacterial ancestry, similar modifications occur, with mammals representing a notable exception where the final product involves the incorporation of the amino acid taurine to form 5-taurinomethyluridine (?m5U34). In bacteria, the lack of these modifications provoked several severe phenotypes notably growth retardation and loss of virulence whereas in human several pathologies such the MELAS, MERRF and others serious diseases are caused by absence of this mitochondrial modification. In addition, the MnmEG complex seems to have a negative impact on iron sulfur cluster assembly of another tRNA modifying enzyme. Our long term project aims at characterizing the structure-function relationship of the two homologues MnmEG from E. coli and its human counterpart the GTPBP3/MTO1 complex. To characterize the enzymes and interrogate their modes of action, we will use traditional biochemical and enzymological approaches in combination with structural methods such as X-ray crystallography and various forms of spectroscopy, as well as small-scale organic synthesis and fast-reaction kinetic methods. In addition we will investigate in vitro and in vivo notably by genetic methods, the new role of MnmEG in the iron sulfur cluster assembly of several enzymes.

Project coordination

Djemel Hamdane (Laboratoire de Chimie des Processus Biologiques)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


CNRS-UMR8229-collège De France Laboratoire de Chimie des Processus Biologiques

Help of the ANR 250,813 euros
Beginning and duration of the scientific project: September 2015 - 42 Months

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