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

Myllykoski, Matti; Kursula, Petri

We also cocrystallized the enzyme with ATP and 3 0 -AMP, which are neither substrates nor products. The fact that they bind the active site can imply that they may be weak inhibitors, and it can be taken as evidence of a general propensity to bind nucleotides in the active site. The LigT orthologue protein PF0027 from Pyrococcus furiosus was shown to require a GTP cofactor for the RNA ligation reaction [3] , while for the E. coli enzyme, such a cofactor has not been reported. Although we attempted crystallization in the presence of GTP as with ATP, no corresponding GTP electron density was found in the resulting crystals (data not shown). Two similar datasets were collected with ATP, one of which has one ATP bound to only one of the 2 monomers in the asymmetric unit. This ATP molecule probably has a partial occupancy and/or some degree of flexibility, as evidenced by residual difference electron density. The major conformation was built in the structure. The other complex has three ATP molecules for two monomers; the space group remains the same. The reason for this fortuituous ambiguity is unknown. In addition to the ligand in both active sites (Fig 2B) , another ATP in the latter crystal form is bound in the vicinity of it in one monomer; this ATP is also involved in crystal contacts. The binding of this second ATP is a strong indication of a propensity for binding further nucleotides in the active site vicinity by E. coli LigT. Residues interacting with the second ATP include Phe159 and Arg6. Again, the α-5 0 -phosphate of the active-site ATP is in the same location as the corresponding phosphate in NADP + , and it is similarly coordinated by Arg6, highlighting putative RNA recognition features extending from the active site. As far as the 3 0 -AMP experiment is concerned, one of the 4 monomers in the asymmetric unit has a bound 3 0 -AMP molecule, with the phosphate group in the same position as in the 2 0 -AMP product complex. Binding of the ligand to the remaining monomers is apparently prevented by crystal contacts. The conformation of 3 0 -AMP differs slightly from that observed for 2 0 -AMP, but the essential recognition features remain the same; stacking of the base against Phe48, binding of the phosphate to the two HxTx motifs in the active site, and C-H. . .π interaction of the sugar ring against Phe8 ( Fig 2C) . As incubation with 2 0 ,3 0 -cAMP resulted in 2 0 -AMP, the 3 0 -AMP complex is irrelevant for the reaction mechanism. To generate 3 0 -AMP in the LigT reaction, the nucleophilic water should attack from the opposite side.