Structural aspects of nucleotide ligand binding by a bacterial 2H phosphoesterase

Myllykoski, Matti; Kursula, Petri

To get further information on the flexibility of the loops, we performed a search for the closest 2H structural homologues (Fig 5B) . The Salami structural homology search detected 11 homologous proteins from the PDB, the best hit representing the previous LigT structure. Vertebrate CNPases were not included in this list, which had 2H proteins from bacteria, viruses, and plants; many of these are annotated as 2 0 -5 0 RNA ligases or phosphoesterases. Superposition of the structures indicated highly similar folding, despite sequence identities as low as 7%, and the largest differences were in the loops mentioned above. This result further highlights the flexibility of the active-site vicinal loops and suggests they may be important in RNA substrate recognition in the entire 2H enzyme family. Visualization of the temperature factors in the LigT crystal structure is in line with this loop flexibility (Fig 5C) . The loop corresponding to the α5-β6 loop is also very flexible in mouse CNPase (Fig 5D) . The dynamics of LigT were also studied using MD simulations. Analysis of the root mean square fluctuations (RMSF) during the simulation (Fig 5E) indicates that the most dynamic segments of the protein correspond to the loops described above, surrounding the active-site cavity. The most dynamic structure appears to be the β hairpin formed between the two C-terminal β strands; interestingly, this hairpin loop is not present in the mammalian CNPase structure. Some loops are more rigid in the simulation than predicted by the crystallographic B factors; this observation may be related to the fact that the reaction product 2 0 -AMP was present in the active site in the simulation run. The results further highlight the flexible nature of the loops, likely to play roles in LigT substrate binding.