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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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Cation dependency of DEER distance distributions, for MnmE mutants S278R1 (left) and E287R1 (right).For each mutant, the left column shows the background corrected dipolar evolution data and the fit obtained by Tikhonov regularization (broken line) and the right column the corresponding distance distribution. The evolution data and the respective distance distributions are colored according to the cation present in the experiment (red, Na+; black, K+; blue, Rb+; green, Cs+; and pale green, NH4+ [only for S278R1, GDP, and GDP-AlFx]). The area under the distance distribution corresponds to the number of interacting spins, derived from the modulation amplitude of the background corrected dipolar evolution data.
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pbio-1000212-g005: Cation dependency of DEER distance distributions, for MnmE mutants S278R1 (left) and E287R1 (right).For each mutant, the left column shows the background corrected dipolar evolution data and the fit obtained by Tikhonov regularization (broken line) and the right column the corresponding distance distribution. The evolution data and the respective distance distributions are colored according to the cation present in the experiment (red, Na+; black, K+; blue, Rb+; green, Cs+; and pale green, NH4+ [only for S278R1, GDP, and GDP-AlFx]). The area under the distance distribution corresponds to the number of interacting spins, derived from the modulation amplitude of the background corrected dipolar evolution data.

Mentions: Previous studies have shown K+ ions to activate the MnmE GTPase. This follows from the finding that dimerization of the MnmE G domains and GDP-AlFx complex formation strictly require K+, which is bound in the dimer interface (Figure 3B), such that its position overlaps with that of an Arg finger required for the GAP-mediated GTP hydrolysis on Ras-like G proteins [20],[22]. Moreover, GTPase activity and AlFx-induced dimerization are at least partially stimulated by cations with an ionic radius comparable to K+ (1.38 Å) such as Rb+ (1.52 Å) and, to a lesser extent, NH4+ (1.44 Å), whereas Na+ (0.99 Å) and Cs+ (1.67 Å) do not show this effect [22]. Consistent with this, Rb+ and NH4+ were also found to be coordinated to the K+ binding site in two MnmE G domain dimer structures GDP complexed with AlFx (pdb 2GJ9 and 2GJA) [22]. To analyze the cation dependency of G domain dimerization in full-length protein in solution, we determined distance distributions for the sites S278R1 and E287R1 in the apo, GDP, GDP-AlFx, and GppNHp bound state in the presence of various cations, i.e., Na+, K+, Rb+, Cs+, and for S278R1 additionally in the presence of NH4+ for the GDP and GDP-AlFx state (Figure 5).


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)

Cation dependency of DEER distance distributions, for MnmE mutants S278R1 (left) and E287R1 (right).For each mutant, the left column shows the background corrected dipolar evolution data and the fit obtained by Tikhonov regularization (broken line) and the right column the corresponding distance distribution. The evolution data and the respective distance distributions are colored according to the cation present in the experiment (red, Na+; black, K+; blue, Rb+; green, Cs+; and pale green, NH4+ [only for S278R1, GDP, and GDP-AlFx]). The area under the distance distribution corresponds to the number of interacting spins, derived from the modulation amplitude of the background corrected dipolar evolution data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749940&req=5

pbio-1000212-g005: Cation dependency of DEER distance distributions, for MnmE mutants S278R1 (left) and E287R1 (right).For each mutant, the left column shows the background corrected dipolar evolution data and the fit obtained by Tikhonov regularization (broken line) and the right column the corresponding distance distribution. The evolution data and the respective distance distributions are colored according to the cation present in the experiment (red, Na+; black, K+; blue, Rb+; green, Cs+; and pale green, NH4+ [only for S278R1, GDP, and GDP-AlFx]). The area under the distance distribution corresponds to the number of interacting spins, derived from the modulation amplitude of the background corrected dipolar evolution data.
Mentions: Previous studies have shown K+ ions to activate the MnmE GTPase. This follows from the finding that dimerization of the MnmE G domains and GDP-AlFx complex formation strictly require K+, which is bound in the dimer interface (Figure 3B), such that its position overlaps with that of an Arg finger required for the GAP-mediated GTP hydrolysis on Ras-like G proteins [20],[22]. Moreover, GTPase activity and AlFx-induced dimerization are at least partially stimulated by cations with an ionic radius comparable to K+ (1.38 Å) such as Rb+ (1.52 Å) and, to a lesser extent, NH4+ (1.44 Å), whereas Na+ (0.99 Å) and Cs+ (1.67 Å) do not show this effect [22]. Consistent with this, Rb+ and NH4+ were also found to be coordinated to the K+ binding site in two MnmE G domain dimer structures GDP complexed with AlFx (pdb 2GJ9 and 2GJA) [22]. To analyze the cation dependency of G domain dimerization in full-length protein in solution, we determined distance distributions for the sites S278R1 and E287R1 in the apo, GDP, GDP-AlFx, and GppNHp bound state in the presence of various cations, i.e., Na+, K+, Rb+, Cs+, and for S278R1 additionally in the presence of NH4+ for the GDP and GDP-AlFx state (Figure 5).

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