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Kissing G domains of MnmE monitored by X-ray crystallography and pulse electron paramagnetic resonance spectroscopy.

Meyer S, Böhme S, Krüger A, Steinhoff HJ, Klare JP, Wittinghofer A - PLoS Biol. (2009)

Bottom Line: Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE.Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle.They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.

ABSTRACT
MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF(x) structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 A. With GDP and AlF(x), which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

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X-ray structures of full-length MnmE dimers.(A) Model of dimeric MnmE obtained from the partial structure of nucleotide-free MnmE from T. maritima, where only the N-terminal domain (NB), but not the helical (HB), or G domain (GB) of molecule B were present in the crystal. The model was obtained by superimposition of molecule A on the N domain of B and the expected positions of the nucleotide binding sites (denoted as NBS) in this model are indicated. (B–D) Ribbon models of X-ray structures of CtMnmE·GDP (B), No MnmE·GDP (C), and Ct MnmE·GppCp (dimer a) (D), with colors of the N, H, and G domains as indicated, and the protomers A and B.
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pbio-1000212-g001: X-ray structures of full-length MnmE dimers.(A) Model of dimeric MnmE obtained from the partial structure of nucleotide-free MnmE from T. maritima, where only the N-terminal domain (NB), but not the helical (HB), or G domain (GB) of molecule B were present in the crystal. The model was obtained by superimposition of molecule A on the N domain of B and the expected positions of the nucleotide binding sites (denoted as NBS) in this model are indicated. (B–D) Ribbon models of X-ray structures of CtMnmE·GDP (B), No MnmE·GDP (C), and Ct MnmE·GppCp (dimer a) (D), with colors of the N, H, and G domains as indicated, and the protomers A and B.

Mentions: The crystal structure of MnmE from Thermotoga maritima reveals a three-domain protein consisting of an N-terminal tetrahydrofolate-binding domain, a central helical domain, and a canonical Ras-like G domain inserted into the helical domain [19]. The asymmetric unit of these crystals contained one MnmE molecule and the N-terminal domain of a second proteolysed MnmE chain interacting with the N-terminal domain of the first molecule, suggesting that MnmE is a dimer in solution (Figure 1A) [19]. By superposition of the first MnmE chain on the second N-terminal domain a model for the full-length homodimer was generated in which the two G domains face each other with a distance of almost 50 Å between the two P-loops (Figure 1A) [19].


Kissing G domains of MnmE monitored by X-ray crystallography and pulse electron paramagnetic resonance spectroscopy.

Meyer S, Böhme S, Krüger A, Steinhoff HJ, Klare JP, Wittinghofer A - PLoS Biol. (2009)

X-ray structures of full-length MnmE dimers.(A) Model of dimeric MnmE obtained from the partial structure of nucleotide-free MnmE from T. maritima, where only the N-terminal domain (NB), but not the helical (HB), or G domain (GB) of molecule B were present in the crystal. The model was obtained by superimposition of molecule A on the N domain of B and the expected positions of the nucleotide binding sites (denoted as NBS) in this model are indicated. (B–D) Ribbon models of X-ray structures of CtMnmE·GDP (B), No MnmE·GDP (C), and Ct MnmE·GppCp (dimer a) (D), with colors of the N, H, and G domains as indicated, and the protomers A and B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749940&req=5

pbio-1000212-g001: X-ray structures of full-length MnmE dimers.(A) Model of dimeric MnmE obtained from the partial structure of nucleotide-free MnmE from T. maritima, where only the N-terminal domain (NB), but not the helical (HB), or G domain (GB) of molecule B were present in the crystal. The model was obtained by superimposition of molecule A on the N domain of B and the expected positions of the nucleotide binding sites (denoted as NBS) in this model are indicated. (B–D) Ribbon models of X-ray structures of CtMnmE·GDP (B), No MnmE·GDP (C), and Ct MnmE·GppCp (dimer a) (D), with colors of the N, H, and G domains as indicated, and the protomers A and B.
Mentions: The crystal structure of MnmE from Thermotoga maritima reveals a three-domain protein consisting of an N-terminal tetrahydrofolate-binding domain, a central helical domain, and a canonical Ras-like G domain inserted into the helical domain [19]. The asymmetric unit of these crystals contained one MnmE molecule and the N-terminal domain of a second proteolysed MnmE chain interacting with the N-terminal domain of the first molecule, suggesting that MnmE is a dimer in solution (Figure 1A) [19]. By superposition of the first MnmE chain on the second N-terminal domain a model for the full-length homodimer was generated in which the two G domains face each other with a distance of almost 50 Å between the two P-loops (Figure 1A) [19].

Bottom Line: Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE.Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle.They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Max-Planck-Institute of Molecular Physiology, Dortmund, Germany.

ABSTRACT
MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlF(x) structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 A. With GDP and AlF(x), which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlF(x) requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.

Show MeSH