Crystallographic structure of an active, sequence-engineered ribonuclease.
X-ray diffraction methods were used to test a synthetic-modeling approach to the sequence engineering of bovine pancreatic ribonuclease. A model of RNase S-peptide (residues 1-20), having a simplified amino acid sequence but retaining elements deduced to be essential for conformation and function, was previously synthesized and found to form a catalytically active and stable complex with native S-protein (residues 21-24). We have now obtained a 3-A-resolution electron density map of this semisynthetic complex which reveals that the conformation of model peptide closely mimics that of native S-peptide, as intended by sequence design. Some small differences from the native structure are observed: Glu-2 and Arg-10 of the model complex are not close enough to form a salt bridge, the position of the His-12 imidazole ring is slightly shifted in the active site, and the peptide's amino terminus is reoriented. Nonetheless, the major structural features predicted to be essential by computer-aided peptide-design analysis are preserved in the model peptide portion of the complex. These include (i) the alpha-helical framework involving residues 3-13, (ii) the catalytically competent orientation of His-12, and (iii) complex-stabilizing non-bonding interactions involving Phe-8 and Met-13 of S-peptide and hydrophobic residues in the cleft region of S-protein. Further, sequence simplification has not introduced any non-native, potentially stabilizing contacts between the model peptide and S-protein. The results emphasize the usefulness, in redesigning native proteins, of categorizing sequence into residues providing conformational framework and those determining intra-and intermolecular surface recognition.