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Abstract
Aminoacyl-tRNA synthetases (aaRSs) are considered as important components in protein translation as they facilitate the attachment of specific transfer RNA (tRNA) to form aminoacyl-tRNAs. Our study focused on understanding the crystal structure of Glutaminyl-tRNA synthetase (GlnRS) from Thermus thermophilus HB8 (PDB ID:5ZDO) and mechanism of formation of enzyme–substrate complex using substrates and its analogs by applying molecular dynamics simulation (MDS) to investigate the conformational changes. Least energy structure of TtGlnRS was considered to dock the enzyme substrates such as glutamine (Gln), glutamic acid (Glu), adenosine monophosphate (AMP), adenosine triphosphate (ATP), QSI and various substrate analogs (2MA, 4SU and 5MU) onto the active site of the enzyme. We focused on comparative analysis of binding specificity between Gln and Glu; similarly, ATP and AMP. Active site organization as observed by MDS analysis showed interactive changes associated with substrate and catalytically important loops. Study found that when tRNAGln specific for GlnRS was docked into the active site of the TtGlnRS enzyme it interacts with 2' OH on the ribose acceptor end of the tRNA. Upon validation with 50 ns MDS, the maximum deviations and conformational changes of secondary structural elements were observed to be high in the loop regions of enzyme–substrate complexes. Binding affinity of ATP to TtGlnRS was further proved by isothermal titration calorimetry.
| Abbreviations | ||
| aaRSs | = | aminoacyl-tRNA synthetases |
| AMP | = | adenosine monophosphate |
| ATP | = | adenosine triphosphate |
| Glide | = | Grid-based LIgand Docking with Energetic |
| GlnRS | = | glutaminyl-tRNA synthetase |
| GRAVY | = | GRand AVerage of hydropathicitY |
| GROMACS | = | GROingen Machine for Chemical Simulations |
| HADDOCK | = | High Ambiguity Driven protein–protein DOCKing |
| ITC | = | isothermal titration calorimetry |
| 2MA | = | 2-methyladenosine 5'-(dihydrogen phosphate) |
| MDS | = | molecular dynamics simulation |
| 5MU | = | 5-methyluridine 5'-monophosphate |
| NPT | = | number of particles, pressure and temperature |
| NVT | = | number of particles, volume and temperature |
| OPLS-AA | = | optimized potential for liquid simulation all atom |
| PDB | = | Brookhaven Protein Databank |
| PME | = | Particle-Mesh Ewald |
| QSI | = | 5'-o-[n-(l-Glutaminyl)-sulfamoyl]adenosine |
| Rg | = | radius of gyration |
| RMSD | = | root mean square deviation |
| RMSF | = | root mean square fluctuation |
| 4SU | = | 4-thiouracil 5'-monophosphate |
| SPC | = | simple point charge |
| tRNA | = | transfer ribo nucleic acid |
| Tt | = | Thermus thermophilus |
| XP | = | extra precision |
Communicated by Ramaswamy H. Sarma
Acknowledgment
MN thanks GE Healthcare, Bangalore for providing permission to utilize the ITC facilities.
Disclosure statement
The authors have no conflict of interest.