Interaction between α-[>>t] conformation and augmented/non-augmented tRNAGlx
Next we looked at the interactions between the α-[>>t ] H8-L -H9 conformation and tRNAGlx with augmented D-helix (A -tRNAGlx) and with non-augmented D-helix (N -tRNAGlx). In the absence of a structure of proteobacterial A -tRNAGlu, the interaction between α-[>>t ] and the D-helix ofA -tRNAGlu was modelled using the structure ofTth -GluRS complexed withTth -A -tRNAGlu (pdb ID: 2cv0). TheEco -GluRS α-[>>t ] motif was structurally superimposed on to the H8-L -H9 motif ofTth -GluRS inTth -GluRS::Tth -A -tRNAGlu complex and interactions of α-[>>t ] with the D-helix of Tth -A -tRNAGlu were analyzed. As shown Figure 5A, the modelled structure exhibited two protein-tRNA H-bonds (D273-G23 and S270-A14). In addition, the negatively charged oxygen atoms of D273 side-chain were favorably placed within interacting distance (4.4 Å) of G22 amino nitrogen atom. This clearly indicated that the α-[>>t ] conformational motif can favorably interact withA -tRNAGlu.
Interactions between α-[>>t ] andN -tRNAGln was also gauged by modeling studies. The N-terminal domain of Eco -GluRS was superimposed on the N-terminal domain of Eco -GlnRS in theEco -GlnRS::Eco -N -tRNAGln complex (Figure 2A) and interactions between α-[>>t ] (Eco -GluRS) andEco -N -tRNAGln were analyzed. As shown in Figure 5B, this resulted in a severe steric clash between G22/G23 ofN -tRNA and D273/E275 of Eco -GluRS. Interactions betweenEco -GluRS α-[>>t ] motif andN -tRNAGln were also analyzed by a different model. In the second model, the template was Tth -GluRS complexed with A -tRNAGlu. Eco -GluRS α-[>>t ] motif was superimposed onTth -GluRS and Eco -N -tRNAGln was superimposed on the Tth -A -tRNAGln. Subsequently, interactions between Eco -GluRS α-[>>t ] andEco -N-tRNAGln were analyzed. Although there were no steric clashes as in the previous case, favorable interactions of D273 with G22 of A -tRNAGlu (seen in Figure 5A) were lost. The two models indicated that the rigid α-[>>t ] conformation would either give rise to steric clashes with N -tRNAGln or lose favorable interactions (as seen withA -tRNAGlu) or both. In other words, the rigid [>t ]-H8-L-H9 conformation seems geared towards discriminating against N-tRNAGln. A key residue involved behind the A -tRNA compatibility and N -tRNA incompatibility of α-[>>t ] is D273, protruding towards tRNA.