Three-dimensional structure of ribonuclease T 1 complexed with guanylyl-2′,5′-guanosine at 1.8 Å resolution

Abstract

The enzyme ribonuclease T 1 (RNase T 1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2′,5′-guanosine (2′,5′-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3σ in the resolution range 6 to 1.8 Å. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T 1/2′,5′-GpG complex and of the near-isomorphous RNase T 1/2′-GMP complex are comparable. The root-mean-square deviation is 0.73 Å between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5′-terminal guanine of 2′,5′-GpG binds to RNase t 1 in much the same way as in the 2′-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3′-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2′,5′-GpG (except the 5′-terminal guanine), which originates from differences in the pucker of the 5′-terminal ribose. The pucker is C2′- exo for the major site ( 2 3 occupancy) and G1′- endo for the minor site ( 1 3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3′-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2′,5′-GpG, which exceeds that of all other inhibitors of type 2′,5′-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca 2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.