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

Myllykoski, Matti; Kursula, Petri

To obtain an insight into larger ligand binding, we carried out small-angle X-ray scattering (SAXS) experiments with samples of LigT and yeast tRNA (Fig 6, Table 2 ). LigT was monomeric in solution, and the obtained 3D shape corresponded closely to that seen in the crystal structure (Chi 2 = 0.8 between experimental SAXS data and calculated data from a LigT monomer). An exception was the highest concentration (>15 mg/ml), which fitted well to a dimeric species; the relevance of this dimerization is not known, and we believe it was an artifact of the very high concentration. tRNA is slightly larger and more elongated than LigT, as expected. A multiphase ab initio modeling approach, taking advantage of the different X-ray scattering properties of RNA and protein, based on SAXS data from the complex and both components alone was employed, and an elongated complex (Fig 6C) fit the experimental data well. The result is a clear indication of the ability of LigT to bind RNA molecules, in this case specifically tRNA. Furthermore, the good fit of the 1:1 complex to the experimental data and the volume of the corresponding ab initio model both imply that the sample was mostly in complex form, with free LigT and tRNA not interfering with SAXS analysis. Whether tRNA would be a true biological substrate or ligand for LigT, is unclear at present, and remains a subject for future work. The fact that tRNA was not observed in the crystal structure can be explained by the commercial yeast tRNA preparation being a mixture of different tRNAs, not homogeneous enough to support crystallization of the complex.