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Category: Microbial Genetics and Molecular Biology
Insights into the GTPase Mechanism of EF-Tu from Structural Studies, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818142/9781555811846_Chap28-1.gif /docserver/preview/fulltext/10.1128/9781555818142/9781555811846_Chap28-2.gifAbstract:
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.
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Conformational rearrangements in EF-Tu, as seen in the X-ray crystal structures of EF-Tu•Mg2+•GppNHp ( Berchtold et al., 1993 ) (a) and EF-Tu•Mg2+•GDP ( Abel et al., 1996 ) (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.
Conformational rearrangements in EF-Tu, as seen in the X-ray crystal structures of EF-Tu•Mg2+•GppNHp ( Berchtold et al., 1993 ) (a) and EF-Tu•Mg2+•GDP ( Abel et al., 1996 ) (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.
A 2F o – F c electron density map in the catalytic center of active EF-Tu, contoured at 1.5 σ 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.
A 2F o – F c electron density map in the catalytic center of active EF-Tu, contoured at 1.5 σ 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.
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.”
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.”
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.
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.
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 β,γ-bridging group, thereby occluding formation of the water chain.
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 β,γ-bridging group, thereby occluding formation of the water chain.
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.
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.
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 β,γ-bridging oxygen (Oβ), 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 β,γ-bridging oxygen (Oβ) of GTP, which would be highly indicative of a dissociative mechanism.
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 β,γ-bridging oxygen (Oβ), 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 β,γ-bridging oxygen (Oβ) of GTP, which would be highly indicative of a dissociative mechanism.