Structural analysis of an RNase T1 variant with an altered guanine binding segment

The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2′-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 Å resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 Å, 2′-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2′-GMP is located about 4.2 Å apart from its position in wild-type ribonuclease T1-2′-GMP complexes, allowing a Ca2+, coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca2+ was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.