Limits...
Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

Show MeSH

Related in: MedlinePlus

Molecular docking of TC-AMP molecule into the active site of Kae1 from P. abyssi. (A1) Active site cavity of Kae1 from P. abyssi with bound AMP-PNP (PDB file 2IVN). The nucleotide is shown as sticks and hydrogen bonds are indicated by dotted lines. (A2) Detailed view of the γ-phosphate binding site. (B1) Active site cavity of Kae1 from P. abyssi with docked TC-AMP. The thermodynamically most favorable conformation (ΔG = −9.7 kcal/mol) is presented. TC-AMP is shown as sticks and hydrogen bonds are indicated by dotted lines. The docking was performed as blind docking by using Autodock Vina 1.1.2 software (see ‘Materials and Methods’ section). B2. Detailed view of the threonyl binding site. Further details are described in the main text.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814370&req=5

gkt720-F6: Molecular docking of TC-AMP molecule into the active site of Kae1 from P. abyssi. (A1) Active site cavity of Kae1 from P. abyssi with bound AMP-PNP (PDB file 2IVN). The nucleotide is shown as sticks and hydrogen bonds are indicated by dotted lines. (A2) Detailed view of the γ-phosphate binding site. (B1) Active site cavity of Kae1 from P. abyssi with docked TC-AMP. The thermodynamically most favorable conformation (ΔG = −9.7 kcal/mol) is presented. TC-AMP is shown as sticks and hydrogen bonds are indicated by dotted lines. The docking was performed as blind docking by using Autodock Vina 1.1.2 software (see ‘Materials and Methods’ section). B2. Detailed view of the threonyl binding site. Further details are described in the main text.

Mentions: The Pa-Kae1 structure in complex with AMP-PNP revealed that the adenine ring makes specific base interactions via its N6 and N1 atoms to the Glu176 and Asn257 side chains. Both O2’ and O3’ hydroxyl groups from the nucleotide ribose moiety are hydrogen-bonded to the Asp159 carboxylate. In addition, the 2’OH group of the ribose moiety interacts with the Gly172 amide group. In the predicted binding of TC-AMP, the nucleoside part of the intermediate could be well superposed with AMP-PNP, such that all of the mentioned interactions were preserved (Figure 6, Supplementary Figure S5). Differences were observed for the binding of the l-threonyl-carbamoyl part of the molecule. The side-chain -OH group of the threonyl moiety interacted with His107 N2, Tyr127 OH, Ser129 OH and the Asp285 carboxylate group. The carboxylate group of the threonyl moiety was contacted by the main chain amide from Gly131, and the α-phosphate (AMP part of the molecule) by the main amide from both Asp285 and Gly253 (and also probably by a Mg2+ ion identified in the Pa-Kae1 structure from residual electronic density next to the iron atom) (Supplementary Figure S5). Interestingly, the side-chain -OH group of threonyl moiety is predicted to participate in the coordination sphere of Fe3+, indicating that iron atom is directly involved in the binding of TC-AMP. Taken together, these data are consistent with the role of Kae1 in binding of TC-AMP intermediate in course of the biosynthesis of t6A modification.Figure 6.


Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification.

Perrochia L, Guetta D, Hecker A, Forterre P, Basta T - Nucleic Acids Res. (2013)

Molecular docking of TC-AMP molecule into the active site of Kae1 from P. abyssi. (A1) Active site cavity of Kae1 from P. abyssi with bound AMP-PNP (PDB file 2IVN). The nucleotide is shown as sticks and hydrogen bonds are indicated by dotted lines. (A2) Detailed view of the γ-phosphate binding site. (B1) Active site cavity of Kae1 from P. abyssi with docked TC-AMP. The thermodynamically most favorable conformation (ΔG = −9.7 kcal/mol) is presented. TC-AMP is shown as sticks and hydrogen bonds are indicated by dotted lines. The docking was performed as blind docking by using Autodock Vina 1.1.2 software (see ‘Materials and Methods’ section). B2. Detailed view of the threonyl binding site. Further details are described in the main text.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814370&req=5

gkt720-F6: Molecular docking of TC-AMP molecule into the active site of Kae1 from P. abyssi. (A1) Active site cavity of Kae1 from P. abyssi with bound AMP-PNP (PDB file 2IVN). The nucleotide is shown as sticks and hydrogen bonds are indicated by dotted lines. (A2) Detailed view of the γ-phosphate binding site. (B1) Active site cavity of Kae1 from P. abyssi with docked TC-AMP. The thermodynamically most favorable conformation (ΔG = −9.7 kcal/mol) is presented. TC-AMP is shown as sticks and hydrogen bonds are indicated by dotted lines. The docking was performed as blind docking by using Autodock Vina 1.1.2 software (see ‘Materials and Methods’ section). B2. Detailed view of the threonyl binding site. Further details are described in the main text.
Mentions: The Pa-Kae1 structure in complex with AMP-PNP revealed that the adenine ring makes specific base interactions via its N6 and N1 atoms to the Glu176 and Asn257 side chains. Both O2’ and O3’ hydroxyl groups from the nucleotide ribose moiety are hydrogen-bonded to the Asp159 carboxylate. In addition, the 2’OH group of the ribose moiety interacts with the Gly172 amide group. In the predicted binding of TC-AMP, the nucleoside part of the intermediate could be well superposed with AMP-PNP, such that all of the mentioned interactions were preserved (Figure 6, Supplementary Figure S5). Differences were observed for the binding of the l-threonyl-carbamoyl part of the molecule. The side-chain -OH group of the threonyl moiety interacted with His107 N2, Tyr127 OH, Ser129 OH and the Asp285 carboxylate group. The carboxylate group of the threonyl moiety was contacted by the main chain amide from Gly131, and the α-phosphate (AMP part of the molecule) by the main amide from both Asp285 and Gly253 (and also probably by a Mg2+ ion identified in the Pa-Kae1 structure from residual electronic density next to the iron atom) (Supplementary Figure S5). Interestingly, the side-chain -OH group of threonyl moiety is predicted to participate in the coordination sphere of Fe3+, indicating that iron atom is directly involved in the binding of TC-AMP. Taken together, these data are consistent with the role of Kae1 in binding of TC-AMP intermediate in course of the biosynthesis of t6A modification.Figure 6.

Bottom Line: We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex.Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA.Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, IFR115, UMR8621-CNRS, 91405 Orsay, France and Université de Lorraine, UMR 1136 INRA/Université de Lorraine Interactions Arbres-Microorganismes, Labex ARBRE, FR EFABA, Faculté des Sciences, 54500 Vandoeuvre, France.

ABSTRACT
N(6)-threonylcarbamoyladenosine (t(6)A) is a universal tRNA modification essential for normal cell growth and accurate translation. In Archaea and Eukarya, the universal protein Sua5 and the conserved KEOPS/EKC complex together catalyze t(6)A biosynthesis. The KEOPS/EKC complex is composed of Kae1, a universal metalloprotein belonging to the ASHKA superfamily of ATPases; Bud32, an atypical protein kinase and two small proteins, Cgi121 and Pcc1. In this study, we investigated the requirement and functional role of KEOPS/EKC subunits for biosynthesis of t(6)A. We demonstrated that Pcc1, Kae1 and Bud32 form a minimal functional unit, whereas Cgi121 acts as an allosteric regulator. We confirmed that Pcc1 promotes dimerization of the KEOPS/EKC complex and uncovered that together with Kae1, it forms the tRNA binding core of the complex. Kae1 binds l-threonyl-carbamoyl-AMP intermediate in a metal-dependent fashion and transfers the l-threonyl-carbamoyl moiety to substrate tRNA. Surprisingly, we found that Bud32 is regulated by Kae1 and does not function as a protein kinase but as a P-loop ATPase possibly involved in tRNA dissociation. Overall, our data support a mechanistic model in which the final step in the biosynthesis of t(6)A relies on a strictly catalytic component, Kae1, and three partner proteins necessary for dimerization, tRNA binding and regulation.

Show MeSH
Related in: MedlinePlus