Structural analysis of an RNase T 1 variant with an altered guanine binding segment

• Published in: Journal of molecular biology Vol. 294; no. 5; pp. 1231 - 1238
• Main Authors: Höschler, Katja, Hoier, Helga, Hubner, Bernd, Saenger, Wolfram, Orth, Peter, Hahn, Ulrich
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 17.12.1999
• Subjects: enzyme specificity; mutagenesis; RNase T 1; substrate recognition; X-ray crystallography; X-ray crystallography; enzyme specificity; RNase T 1; mutagenesis; substrate recognition; wt
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.3324

The ribonuclease T 1 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 T 1-2′-GMP complexes, allowing a Ca 2+, 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 Ca 2+ was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.