Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering.
Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.
Hakoshima
T
T
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Toda
S
S
Sugio
S
S
Tomita
K
K
Nishikawa
S
S
Morioka
H
H
Fuchimura
K
K
Kimura
T
T
Uesugi
S
S
Ohtsuka
E
E
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000235:genetics; D005658:Fungi / Q000201:enzymology; D006147:Guanine; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D006163:Ribonuclease T1 / Q000235:genetics; D014961:X-Ray Diffraction
D006147:Guanine; D004722:Endoribonucleases; D006163:Ribonuclease T1