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Chapter 28 : Insights into the GTPase Mechanism of EF-Tu from Structural Studies

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Insights into the GTPase Mechanism of EF-Tu from Structural Studies, Page 1 of 2

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Abstract:

The main role of EF-Tu is clearly in the elongation phase of bacterial protein synthesis. The protein transports aminoacylated tRNA (aa-tRNA) molecules to the programmed ribosome and profoundly contributes to an accurate and fast translation of mRNAs into proteins. EF-Tu was the first protein found to be regulated by the binding and subsequent hydrolysis of GTP, making it a paradigm for the superfamily of regulatory GTPases. The rather loose structure of the GDP complex, originally seen with an EF-Tu that had been proteolytically cleaved at at least two sites, was later validated by X-ray analysis of crystals of intact EF-Tu·GDP. While the mechanism of the ribosome-mediated GTPase reaction of EF-Tu is thus entirely unclear, very little is known about the intrinsic GTP-hydrolyzing activity exhibited by the enzyme in the absence of the ribosome. In the amino acid sequence of EF-Tu, the conserved glutamine residue of Gα and Ras is replaced by His-85. The importance of the hydrophobic gate in protecting the nucleophilic water molecule from premature activation was tested by replacing the wing residues (V20S and I61A mutants). Both mutants do show a somewhat elevated intrinsic GTPase, but instead of His-85 swinging in to activate the nucleophilic water, the authors observe the rearrangement of a chain of water molecules extending from bulk solvent to the γ-phosphate.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28

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Elongation Factor Tu
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Ribosome Binding Site
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Figures

Image of Figure 1
Figure 1

Conformational rearrangements in EF-Tu, as seen in the X-ray crystal structures of EF-Tu•Mg2+•GppNHp ( ) (a) and EF-Tu•Mg2+•GDP ( ) (b). Global rearrangements are effected by two mobile structural elements, the switch I (residues 40 to 62) and switch II (residues 80 to 100) regions, which are highlighted in dark and medium shading, respectively. The nucleotide and Mg2+ ion are rendered in diagrams for each structure.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 2
Figure 2

A 2F – F electron density map in the catalytic center of active EF-Tu, contoured at 1.5 above the mean. The nucleophilic water (wat) molecule (411) is shielded from His-85 and bulk solvent by the hydrophobic side chains of Val-20 and Ile-61, the so-called hydrophobic gate.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 3
Figure 3

Alternative mechanistic pathway extremes for phosphoryl transfer reactions, including the GTP hydrolysis reaction in EF-Tu. A fully dissociative reaction (top) is characterized by a loss of negative charge on the transferred metaphosphate. The analogous phosphoryl moiety exhibits a net increase in negative charge in an associative transition state (bottom). GDP is denoted by “OR.”

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 4
Figure 4

Schematic drawing illustrating the interaction between His-85 and His-119 in EF-Tu•GDP, which is interrupted by insertion of a phenylalanine from the nucleotide exchange factor in the EF-Tu•EF-Ts complex.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 6
Figure 6

Schematic illustration of the alternative modes of interaction between Asp-21 and GppNHp. (a) In the open binding mode, the side chain of Asp-21 interacts with waters (wat) 473 and 499 of the water chain connecting the γ-phosphate to bulk solvent. (b) In the closed binding mode, the side chain of Asp-21 moves to accept a hydrogen bond from the ,γ-bridging group, thereby occluding formation of the water chain.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 5
Figure 5

Conformational variability in the nucleotide binding pocket is observed for residues 20 to 22 of the phosphate binding loop (P loop) in the EF-Tu complex with GppNHp and manifested to the largest extent in the side chain of Asp- 21. Waters 473 and 499 are only present in one of the two conformations of the P loop. Shown is a 2Fo –Fc electron density map contoured at 1.2 σ above the mean.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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Image of Figure 7
Figure 7

Possible pre-transition state pathways for dissociative GTP hydrolysis catalyzed by EF-Tu. (a) Two-water mechanism involving protonation of the γ-phosphate through the water chain prior to nucleophilic attack by the active-site water, 411. Very likely, the proton originating from the water chain is immediately transferred to the ,γ-bridging oxygen (O), thereby facilitating the dissociation of the leaving group. (b) General-acid-catalyzed hydrolysis of GTP. Asp-21 transfers a proton from the proximal water molecule (473) of the water channel directly to the ,γ-bridging oxygen (O) of GTP, which would be highly indicative of a dissociative mechanism.

Citation: Hilgenfeld R, Mesters J, Hogg T. 2000. Insights into the GTPase Mechanism of EF-Tu from Structural Studies, p 347-357. In Garett R, Douthwaite S, Liljas A, Matheson A, Moore P, Noller H (ed), The Ribosome. ASM Press, Washington, DC. doi: 10.1128/9781555818142.ch28
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