We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2′AMP and 2′UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2′GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A ̊, b = 46.71 A ̊, c = 41.02 A ̊ for the complex with 2′AMP and a = 48.97, b = 46.58 A ̊, c = 40.97 A ̊ for the complex with 2′UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2′AMP and 2′UMP were collected on a diffractometer at 1.7 Å and 2.4 Å resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10-3m, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2′GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme. © 1990.
