Crystal structure of sulfolobus tokodaii sua5 complexed with L‐threonine and AMPPNP

Abstract

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N(6)-threonylcarbamoyl moiety is composed of L-threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L-threonine-binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L-threonine more strongly than L-serine and glycine. The Kd values of Sua5 for L-threonine and L-serine are 9.3 μM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L-threonine, at 1.8 Å resolution. The L-threonine is bound next to AMPPNP in the same pocket of the N-terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine-specific recognition. The carboxyl group and the side-chain hydroxyl and methyl groups of L-threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L-threonine is located in a suitable position to react together with ATP for the synthesis of N(6)-threonylcarbamoyladenosine.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…The binding of ATP could require the binding of L-threonine to occur first, providing the contact surface area for ATP to bind in the correct conformation. As seen in the Sua5 structure (50), contacts are observed between ATP and the L-threonine with the L-threonine buried further into the Sua5 binding cavity (Fig. 7B).…”

“…L-Threonine-binding Site-E. coli TsaC and S. tokodaii Sua5 have been shown to exhibit specificity for L-threonine over other amino acids (25,50), and E. coli TsaC has been shown to catalyze TC-AMP formation (17). Therefore, we sought to characterize the TsaC/L-threonine binding interface to provide insight into structural aspects of the enzymatic mechanism.…”

“…In the co-crystal structure of S. tokodaii Sua5 with L-threonine, the corresponding residues, Thr-34 and Ser-182, both interact with L-threonine (50). S. tokodaii Sua5 Thr-34 forms a hydrophobic environment for the ␥-carbon of L-threonine, and Ser-182 forms two hydrogen bonds with the carboxyl oxygen (50). From our molecular docking studies, we observe Ser-176 coordinating L-threonine in a similar fashion.…”

“…9). These residues have been shown to surround the adenine-binding site in co-crystal structures of both E. coli HypF (middle domain, residues 188 -378) and S. tokodaii Sua5 with the nonhydrolysable ATP analog AMP-PNP (30,50). For HypF, the adenine is buried in a hydrophobic environment between Leu-277 and Pro-249, and it forms weak hydrogen bonds with Glu-296 and Arg-372 (30).…”

“…Pro-249 near the ATP-binding site in HypF is not conserved in this family of proteins and is not present in TsaC. Sua5 coordinates the adenosine with hydrophobic residues Ile-66, Val-101, Ala-120, and Ile-184 (50). When TsaC is aligned with the HypF and Sua5 structures, Ile-59, Ala-115, and Arg-188 (resonances that were all greatly affected during the titration with ATP) correspond to the residues in the other two proteins that are important for the binding of the adenosine.…”