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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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DEER characterization of nucleotide-dependent domain movements of MTSSL labeled MnmE (E287R1, D366R1, S278R1, and I105R1).(A) Left panel, background corrected dipolar evolution data for the apo, GDP, GppNHp, and GDP-AlFx state of the respective MnmE mutants as indicated. Centre panel: dipolar spectra (Fourier transformation of the dipolar evolution data in the left panel). Right column: distance distributions obtained by Tikhonov regularization. All plots are normalized by amplitude. Broken lines in the left and center panel are fits to the data obtained by Tikhonov regularization. For S278R1 in apo, GDP, and GDP-AlFx state, alternative fits and resulting distance distributions obtained with smaller regularization parameters α, are shown in corresponding pale colours. (B) Data for S278R1 in the GDP-, GppNHp-, and GDP-AlFx state analyzed assuming a sum of Gaussian distributed conformers. Left panel: background corrected dipolar evolution data. Centre panel: goodness-of-fit (χ2) surfaces, created by random sampling of distance and width for each Gaussian population in the distance distributions shown in the right panel. Plots in the left and right column are normalized by amplitude. Broken lines in the left panel are fits to the data.
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pbio-1000212-g004: DEER characterization of nucleotide-dependent domain movements of MTSSL labeled MnmE (E287R1, D366R1, S278R1, and I105R1).(A) Left panel, background corrected dipolar evolution data for the apo, GDP, GppNHp, and GDP-AlFx state of the respective MnmE mutants as indicated. Centre panel: dipolar spectra (Fourier transformation of the dipolar evolution data in the left panel). Right column: distance distributions obtained by Tikhonov regularization. All plots are normalized by amplitude. Broken lines in the left and center panel are fits to the data obtained by Tikhonov regularization. For S278R1 in apo, GDP, and GDP-AlFx state, alternative fits and resulting distance distributions obtained with smaller regularization parameters α, are shown in corresponding pale colours. (B) Data for S278R1 in the GDP-, GppNHp-, and GDP-AlFx state analyzed assuming a sum of Gaussian distributed conformers. Left panel: background corrected dipolar evolution data. Centre panel: goodness-of-fit (χ2) surfaces, created by random sampling of distance and width for each Gaussian population in the distance distributions shown in the right panel. Plots in the left and right column are normalized by amplitude. Broken lines in the left panel are fits to the data.

Mentions: Figure 4A illustrates the results of the DEER measurements in the presence of 100 mM KCl, where the left panel shows the background-corrected dipolar evolution data, the centre panel the respective dipolar spectra, and the right panel the corresponding distance distributions (obtained by Tikhonov regularization; see Methods), which are summarized in Table 2. The DEER analysis of mutant E287R1 (R1 denotes the MTSSL side chain), close to the top of the G domain in Gα2, indicates one major peak centered at a distance of 55 Å for the apo- and 53 Å for the GDP-bound state. This distances correspond well to the Cβ-Cβ distances in the TmMnmE crystal structure model of 53 Å (the corresponding residues in the CtMnmE and NoMnmE structures are not resolved) and is therefore in agreement with an open conformation of the G domains. For D366R1 (situated at Gα6), a well-defined interspin distance distribution centered at 67 Å in the apo- state and 65 Å in the GDP-bound state could be observed in good agreement with the distances obtained from the TmMnmE dimer model (62 Å) and NoMnmE·GDP (63 Å), suggesting again an open conformation of the G domains. The corresponding Cβ-Cβ distance in CtMnmE·GDP dimer is somewhat shorter (57 Å), which is due to the different orientation of G domains in this structure (Figure 2A) and to the different tilting of Gα6 (Figure 2B). From the E287R1 and D366R1 data in the apo- and GDP-bound states, we conclude that instead of a continuum of freely moving orientations, the MnmE G domains seem to have defined major orientation reflected by the distance distributions.


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)

DEER characterization of nucleotide-dependent domain movements of MTSSL labeled MnmE (E287R1, D366R1, S278R1, and I105R1).(A) Left panel, background corrected dipolar evolution data for the apo, GDP, GppNHp, and GDP-AlFx state of the respective MnmE mutants as indicated. Centre panel: dipolar spectra (Fourier transformation of the dipolar evolution data in the left panel). Right column: distance distributions obtained by Tikhonov regularization. All plots are normalized by amplitude. Broken lines in the left and center panel are fits to the data obtained by Tikhonov regularization. For S278R1 in apo, GDP, and GDP-AlFx state, alternative fits and resulting distance distributions obtained with smaller regularization parameters α, are shown in corresponding pale colours. (B) Data for S278R1 in the GDP-, GppNHp-, and GDP-AlFx state analyzed assuming a sum of Gaussian distributed conformers. Left panel: background corrected dipolar evolution data. Centre panel: goodness-of-fit (χ2) surfaces, created by random sampling of distance and width for each Gaussian population in the distance distributions shown in the right panel. Plots in the left and right column are normalized by amplitude. Broken lines in the left panel are fits to the data.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749940&req=5

pbio-1000212-g004: DEER characterization of nucleotide-dependent domain movements of MTSSL labeled MnmE (E287R1, D366R1, S278R1, and I105R1).(A) Left panel, background corrected dipolar evolution data for the apo, GDP, GppNHp, and GDP-AlFx state of the respective MnmE mutants as indicated. Centre panel: dipolar spectra (Fourier transformation of the dipolar evolution data in the left panel). Right column: distance distributions obtained by Tikhonov regularization. All plots are normalized by amplitude. Broken lines in the left and center panel are fits to the data obtained by Tikhonov regularization. For S278R1 in apo, GDP, and GDP-AlFx state, alternative fits and resulting distance distributions obtained with smaller regularization parameters α, are shown in corresponding pale colours. (B) Data for S278R1 in the GDP-, GppNHp-, and GDP-AlFx state analyzed assuming a sum of Gaussian distributed conformers. Left panel: background corrected dipolar evolution data. Centre panel: goodness-of-fit (χ2) surfaces, created by random sampling of distance and width for each Gaussian population in the distance distributions shown in the right panel. Plots in the left and right column are normalized by amplitude. Broken lines in the left panel are fits to the data.
Mentions: Figure 4A illustrates the results of the DEER measurements in the presence of 100 mM KCl, where the left panel shows the background-corrected dipolar evolution data, the centre panel the respective dipolar spectra, and the right panel the corresponding distance distributions (obtained by Tikhonov regularization; see Methods), which are summarized in Table 2. The DEER analysis of mutant E287R1 (R1 denotes the MTSSL side chain), close to the top of the G domain in Gα2, indicates one major peak centered at a distance of 55 Å for the apo- and 53 Å for the GDP-bound state. This distances correspond well to the Cβ-Cβ distances in the TmMnmE crystal structure model of 53 Å (the corresponding residues in the CtMnmE and NoMnmE structures are not resolved) and is therefore in agreement with an open conformation of the G domains. For D366R1 (situated at Gα6), a well-defined interspin distance distribution centered at 67 Å in the apo- state and 65 Å in the GDP-bound state could be observed in good agreement with the distances obtained from the TmMnmE dimer model (62 Å) and NoMnmE·GDP (63 Å), suggesting again an open conformation of the G domains. The corresponding Cβ-Cβ distance in CtMnmE·GDP dimer is somewhat shorter (57 Å), which is due to the different orientation of G domains in this structure (Figure 2A) and to the different tilting of Gα6 (Figure 2B). From the E287R1 and D366R1 data in the apo- and GDP-bound states, we conclude that instead of a continuum of freely moving orientations, the MnmE G domains seem to have defined major orientation reflected by the distance distributions.

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
Related in: MedlinePlus