Comparative molecular modeling and crystallization of P‐30 protein: A novel antitumor protein of Rana pipiens oocytes and early embryos

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Abstract
The P‐30 protein (Onconase™)1 of Rana pipiens oocytes and early embryos is homologous to members of the pancreatic ribonuclease superfamily and exhibits an antitumor activity in vitro and in vivo. It appears that the ribonucleolytic activity of P‐30 protein may be required for its antitumor effects. A comparative molecular model of P‐30 protein has been constructed based upon the known three‐dimensional structure of bovine pancreatic RNase A in order to provide structural information. Functionally, these enzymes hydrolyze oligoribonucleotides to pyrimidine‐3′‐phosphate monoesters and 5′‐OH ribonucleotides. In the modeling procedure, automated sequence alignments were revised based upon the inspection of the RNase A structure before the amino acids of the P‐30 protein were assigned the coordinates of the RNase A template. The inevitable intermolecular steric clashes that result were relieved on an interactive graphics device through theadjustment of side chain torsion angles. This process was followed by energy minimization of the model, which served to optimize stereochemical geometry and to relieve any remaining unacceptably close contacts. The resulting model retains the essential features of RNase A as sequence insertions and deletions are almost exclusively found in exposed surface loops. The all atom superposition of active site residues of the P‐30 protein model and an identically minimized RNase A structure has a root mean square deviation of 0.52 Å. Though tentative, the model is consistent with a pyrimidine specificity. Further, the model suggests Lys9 (P‐30 protein) can donate a hydrogen bond to the active site phosphate, whereas it is unlikely that P‐30 protein binds the 3′‐ribonucleotide in a fashion similar to RNase A. P‐30 protein has been crystallized in an orthorhombic space group, P212121, with unit cell dimensions, a = 40.76, b = 69.77, and c = 32.54 Aring;. The crystals grow as small rosettes from an ammonium sulfate solution at pH 4.5.