Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR

Abstract

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.

Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

“…This is in agreement with the role of the histidine residue as a regulatory or catalytic element in the active site of this protein (Berchtold et al, 1993;Cool and Parmeggiani, 1991). A lipophilic leucine side chain not containing a proton-donating or proton-accepting functional group cannot fulfill the function ascribed to residue 85 or its equivalent in regulatory GTPases (Schweins et al, 1995;Coleman et al, 1994;Frech et al, 1994;Sondek et al, 1994;Limmer et al, 1992;PrivC et al, 1992;Pai et al, 1990).…”

Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

“…This is in agreement with the role of the histidine residue as a regulatory or catalytic element in the active site of this protein (Berchtold et al, 1993;Cool and Parmeggiani, 1991). A lipophilic leucine side chain not containing a proton-donating or proton-accepting functional group cannot fulfill the function ascribed to residue 85 or its equivalent in regulatory GTPases (Schweins et al, 1995;Coleman et al, 1994;Frech et al, 1994;Sondek et al, 1994;Limmer et al, 1992;PrivC et al, 1992;Pai et al, 1990).…”

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

“…His-84 of EF-Tu has been proposed to activate a water molecule for nucleophilic attack on the ␥-phosphate by extracting one of its protons (3,4). This proposal agrees with studies identifying His-84 as the catalytic residue that stabilizes the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or the ␥-phosphate group of GTP (5).…”

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

“…is deshielded, as expected for participation in a hydrogen bond, with a chemical shift of 8H = 10.4; a chemical shift of 6H = 9.2 was suggested for this amide proton in the Ras-GTP[-yS] complex (64)(65)(66)(67)(68). Analogous downfield amide proton resonances with bound GDP and GTP have also been suggested for EF-Tu, which is homologous to Ras (69).…”

Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

“…This is in agreement with the role of the histidine residue as a regulatory or catalytic element in the active site of this protein (Berchtold et al, 1993;Cool and Parmeggiani, 1991). A lipophilic leucine side chain not containing a proton-donating or proton-accepting functional group cannot fulfill the function ascribed to residue 85 or its equivalent in regulatory GTPases (Schweins et al, 1995;Coleman et al, 1994;Frech et al, 1994;Sondek et al, 1994;Limmer et al, 1992;PrivC et al, 1992;Pai et al, 1990).…”

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

“…His-84 of EF-Tu has been proposed to activate a water molecule for nucleophilic attack on the ␥-phosphate by extracting one of its protons (3,4). This proposal agrees with studies identifying His-84 as the catalytic residue that stabilizes the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or the ␥-phosphate group of GTP (5).…”

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

“…is deshielded, as expected for participation in a hydrogen bond, with a chemical shift of 8H = 10.4; a chemical shift of 6H = 9.2 was suggested for this amide proton in the Ras-GTP[-yS] complex (64)(65)(66)(67)(68). Analogous downfield amide proton resonances with bound GDP and GTP have also been suggested for EF-Tu, which is homologous to Ras (69).…”

Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu

“…This is in agreement with the role of the histidine residue as a regulatory or catalytic element in the active site of this protein (Berchtold et al, 1993;Cool and Parmeggiani, 1991). A lipophilic leucine side chain not containing a proton-donating or proton-accepting functional group cannot fulfill the function ascribed to residue 85 or its equivalent in regulatory GTPases (Schweins et al, 1995;Coleman et al, 1994;Frech et al, 1994;Sondek et al, 1994;Limmer et al, 1992;PrivC et al, 1992;Pai et al, 1990).…”

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

“…His-84 of EF-Tu has been proposed to activate a water molecule for nucleophilic attack on the ␥-phosphate by extracting one of its protons (3,4). This proposal agrees with studies identifying His-84 as the catalytic residue that stabilizes the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or the ␥-phosphate group of GTP (5).…”

Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

“…is deshielded, as expected for participation in a hydrogen bond, with a chemical shift of 8H = 10.4; a chemical shift of 6H = 9.2 was suggested for this amide proton in the Ras-GTP[-yS] complex (64)(65)(66)(67)(68). Analogous downfield amide proton resonances with bound GDP and GTP have also been suggested for EF-Tu, which is homologous to Ras (69).…”