Docking models of GluRS and tRNA

Based on the observation that the non-discriminating MtGluRS and the discriminating TtGluRS carry both the same arginine supposed to be able to discriminate between tRNA^Glu and tRNA^Gln [Sekine S et al., 2001] docking models of both GluRSs with both tRNAs were constructed. This was done in order to validate the mechanism of discrimination with the two different anticodons of tRNA^Glu (CUC) and tRNA^Gln (CUG) (Fig. 5-8). The conformations of MtGluRS in complexes with tRNA^Glu and tRNA^Gln were modelled on the basis of the experimental structures of complexes of TtGluRS with tRNA^Glu (1N77). To model the tRNA-bound form of MtGluRS the TtGluRS enzyme in chain A from the PDB entry 1N77 served as a template. In the case of TtGluRS no major conformational differences were observed between the tRNA-bound and free enzyme [Sekine S et al., 2003]. Binding of the tRNA molecule mainly involved minor rotations (~ 6-8°) of single domains without changes in their fold.

Similar can be expected for MtGluRS and therefore the tRNA-bound state was modelled manually by adapting the relative domain orientations as observed for TtGluRS-tRNA^Glu. Starting models of M. tuberculosis tRNA^Glu (CUC) and tRNA^Gln (CUG) were also built manually using the modelling functions of COOT. As a template for the models the tRNA^Glu structure from T. thermophilus (1N77; chain C) was used. Prior to the modelling the secondary structure of Mt-tRNA^Glu and Mt-tRNA^Gln was predicted with the program tRNAscan-SE [Lowe TM and Eddy SR, 1997] provided by the Genomic tRNA data base services⁷. Both enzyme and tRNAs were energy minimized prior to docking. After minimization tRNAs and the enzyme were docked by superimposing both onto one TtGluRS-tRNA^Glu complex from 1N77 (chains A and C). For superposition of the enzyme and the tRNA Cα and phosphate atoms were used, respectively. Finally the contacts at the enzyme-tRNA interfaces were manually adjusted on the basis of contacts conserved between tRNA and enzyme in both TtGluRS and MtGluRS. The completed complex structures were refined and validated with COOT. Ramachandran plots for the docking complexes showed all non-glycine and non-proline residues in allowed regions.

7 http://lowelab.uscsc.edu/GtRNAdb

All theoretically built models of the tRNAs were further validated using the software X3DNA [Lu XJ and Olson WK, 2003]. Results for the geometry of the modelled tRNAs were compared to those from the tRNA^Glu model of obtained by crystallography (PDB-ID 1N77). Upon this comparison the geometries of the theoretically modelled tRNA^Glu/Gln for M. tuberculosis were found to be in good agreement with the crystallographic data for tRNA^Glu from T. thermophilus.

Subsequently the docking models of GluRS-tRNA^Glu/Gln were submitted to energy minimization and the resulting models were found to have good geometries.

Differences in the backbone conformations between MtGluRS-tRNA^Glu and TtGluRS-tRNA^Glu are only observed for insertions or deletions in loop segments. In the docking complex MtGluRS-tRNA^Glu the enzyme mainly interacts with the acceptor arm, the augmented D-helix and the anticodon arm. The latter is contacted by the KLSKR loop

Figure 5-8: Cloverleaf representation of tRNA^Gln (left) and tRNA^Glu (right) for M. tuberculosis (A) and T. thermophilus (B). The anticodon is indicated by red boxes. The acceptor arm is highlighted in grey, the D-stem and variable loop in dark grey and the anticodon loop in light grey B

A

as well as by the helices α4 and α5. Both helices show elevated backbone temperature factors (averaged 32 Ų compared to 21 Ų for the domains RF, SC and AB 4) in the MtGluRS apo structure. Thereby the helices indicate conformational flexibility that might facilitate binding of the acceptor arm upon tRNA binding. The conformations of the helices α4 and α5 are similar in the tRNA^Glu complexes with both MtGluRS and TtGluRS. Additionally MtGluRS makes contact to the phosphate group of the nucleotides G70 and C71 via the KLSKR loop in the same way the KISKR loop of TtGluRS is involved into contacts with the phosphates of the corresponding nucleotides G70 and U71. These interactions are involved in the rearrangement of the active site upon tRNA^Glu binding [Sekine S. et al., 2003].

Further similarities in tRNA^Glu binding between MtGluRS and TtGluRS include contacts of residues of the SC domain to the D stem. Especially contacts of the

nucleotides 11 to 13 in MtGluRS-tRNA^Glu to the D stem are similar to those observed in the TtGluRS-tRNA^Glu-ATP structure. Here these contacts were reported to play a role in the tRNA dependent switching of the ATP binding mode [Sekine S. et al., 2003]. In MtGluRS-tRNAGlu the loop 291 to 298 is close to the minor groove of the D arm involving base specific contacts for Asp293, Asp296 and Lys323. Here the base specific contact of Asp296 corresponds to the essential direct interaction seen in TtGluRS-tRNA^Glu between Glu282 and the tRNA [Nureki O et al., 1995]. In the MtGluRS-tRNA^Glu model a further specific interaction between Asp293 and G22 is observed. In total, close similarity in the contacts can be observed in both

GluRS-Figure 5-9: Anticodon recognition in the modelled MtGluRS-tRNA^Glu (A) and MtGluRS-tRNA^Gln (B) complexes. See text for details

B

tRNA^Glu complexes from M. tuberculosis and T. thermophilus. For both complexes all conserved residues in the contact areas between enzyme and tRNA are observed in closely similar conformations.