The H8-loop-H9 in Eco-GluRS as a tRNAGln-discriminatory feature
Having determined the structure of Eco -GluRS, we compared the sequence and structural features of its N-terminal catalytic domain with that of tRNAGln-bound Eco -GlnRS (PDB Id: 1gts). Being homologous, the overall folds of the two catalytic domains are similar. However, the structural superimposition brought out some important differences. Specifically, we looked at amino acid stretches in the two proteins at the binding interface of the D-helix region of tRNAGln, focussing on differences, since it must be this region that differentially interacts with the uniquely different D-helix regions of tRNAGlu and tRNAGln, triggering tRNAGlx-specificity. The analysis identified two stretches (Figure 2), residues 303-335 in Eco -GlnRS and residues 257-311 in Eco -GluRS, both end-capped with helices (Helix 11 and Helix 12 in GlnRS; Helix 8 and Helix 10 in GluRS). While the two terminal helices are well superposed in both, the intervening stretches are not. The ~10 residue inter-helical stretch in GlnRS assumes an extended structure and is proximal to the tRNAGln D-helix. In contrast, the inter-helical stretch in GluRS is almost three times longer, of which the conformation and tRNAGln proximity of 10 residue stretch at the C-terminal are similar to the inter-helical stretch in GlnRS. However, conformation adopted by the first 20 residues (towards the N-terminal) in GluRS has no counterpart in GlnRS. This stretch contains a helix (Helix 9) in GluRS with no counterpart in GlnRS. In addition, it contains a loop between Helix 8 and Helix 9 that is at the interface of the D-helix of tRNAGln. In other words, GluRS exhibits a unique [Helix 8]-[loop]-[Helix 9] (H8-L -H9) motif situated at the tRNAGln D-helix interface that is absent in GlnRS.