tRNAGln-discriminatory status of proteobacterial GluRSs in outlier PCA cluster D
PCA performed on H8-L -H9 proteobacterial motifs resulted in three clusters, of which cluster D was more towards the tRNA-nondiscriminatory clusters along PC1. Two members of cluster D have been discussed: (i) HPY(T1), which was designated asN -tRNAGln-discriminatory based on its dymanic α-[>>t ] conformation and (ii) PAE(D+), which was designated asN -tRNAGln-discriminatory based on its dynamic β-[>>t ] conformation. Here we closely examine sequence and structural patterns of all members of cluster D. In addition to PAE, cluster D contains 13 more D(+)-GluRSs from the γ-proteobacterial class (TTU, MMW, CSA, HEL, ABO, CJA, MAQ, HCH, PAR, SDE, AVN, ACI and MCT) whose H8-L-H9 sequences are very similar to that of Pae -GluRS (Figure S10), all associated withA -tRNAGlu andN -tRNAGln. Since Pae -GluRS displayed the β-[>>t]-H8-L -H9 motif, compatible with both A -tRNAGlu and incompatible withN -tRNAGln, we predict all 13 D(+)-GluRSs to be tRNAGln-non-discriminatory.
Cluster D contained 5 D(+)-GluRSs from the δ-proteobacterial class (HOH, DPS, DAK, DPR and BBA). Although the H8-L -H9 loop length in these were one residue longer than Eco -GluRS, interestingly, all except BBA displayed G-(D/T)-D sequence motif at its center (Figure S11A). In absence of experimental structures we assessed the loop conformations of all 5 GluRSs using AlphaFold models. As shown in Figure S11B, except BBA (that lacked the central G-(D/T)-D sequence motif) all H8-L-H9 motifs assumed the [>>t] conformation where the side-chain of the second Asp residue in G-(D/T)-D protruded out of the tip of the loop overlapping well with D273 of Eco -GluRS. Interestingly, like other [>>t] conformations encountered so far, the loop did not shown α- or β-turns; to emphasize this point we name this conformation as ε-[>>t] (13-residue loop with no α-/β-turns in the loop). A summary of all types of [>>t] conformations identified so far is shown in Figure 7.
The presence of the N -tRNAGlx-discriminating ε-[>>t] conformation implies thatHoh -GluRS (with A -tRNAGln1 andA -tRNAGln2) will be tRNAGln-non-discriminatory, Dps -GluRS (withN -tRNAGln1) will be tRNAGln-discriminatory, Dak -GluRS and andDpr -GluRS (both with N -tRNAGln1 andA -tRNAGln2), on the other hand, will be tRNAGln2-non-discriminatory but tRNAGln1-discriminatory. Bba -GluRS (with the away from tRNA conformation [t>> ] and N -tRNAGln1) will beN -tRNAGln-non-discriminatory.