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 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 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 A apart from its position in wild-type ribonuclease T1-2'-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.
Höschler
K
K
Institut für Biochemie Fakultät für Biowissenschaften Pharmazie und Psychologie, Universität Leipzig, Talstrasse 33, Leipzig, 04103, Germany.
Hoier
H
H
Hubner
B
B
Saenger
W
W
Orth
P
P
Hahn
U
U
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D014644:Genetic Variation / Q000235:genetics; D006147:Guanine / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D006163:Ribonuclease T1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D010710:Phosphates; D014867:Water; C026693:2'-guanylic acid; D006147:Guanine; D012313:RNA; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1; D002118:Calcium