Cytotoxic ribonuclease variants
This invention relates to cytotoxic variants of human ribonuclease 1 (RNase 1) identified through analysis of the interaction between RNase 1 and the human ribonuclease inhibitor (hRI) as defined by the three dimensional (3-D) atomic structure of the RNase1 hRI complex. Also disclosed is the 3-D structure of the hRI•RNase 1 complex and methods for designing the RNase 1 variants.
1. A method of modifying human Ribonuclease (RNase 1) to make an engineered cytotoxic RNase 1 polypeptide, the method comprising the steps of:
(a) identifying electrostatic anchor amino acid residues in the three dimensional crystal structure of the human Ribonuclease Inhibitor•human Ribonuclease (hRI•RNase 1) complex defined by atomic coordinate data deposited in the Protein Data Bank (PDB) as PDB Accession No. 1Z7X and set forth in Appendix 1 of U.S. Pat. No. 7,655,757, wherein RNase 1 is defined by SEQ ID NO:2 and hRI is defined by SEQ ID NO: 4; and
(b) modifying the anchor amino acid residues of RNase 1 identified in step (a) to make an engineered cytotoxic RNase 1 polypeptide, which inhibits binding to hRI through electrostatic repulsion and/or steric hindrance.
2. The method of claim 1 wherein the electronic anchor residues are located at positions 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 111 or 118 of SEQ ID NO:2.
3. The method of claim 1 wherein the electronic anchor residues are located at positions 85 to 94 and at positions 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111, or 118 of SEQ ID NO:2.
4. The method of claim 1 wherein the electronic anchor residues are involved in electrostatically repelling and/or sterically hindering binding of RI to the engineered RNase 1.
5. The method of claim 1 wherein the engineered RNase 1 retains ribonucleolytic activity relative to SEQ ID NO:2.
6. The method of claim 1 wherein the engineered RNase 1 exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2.
7. The method of claim 1 wherein the engineered RNase 1 exhibits a lower binding affinity for hRI than SEQ ID NO:2.
8. The method of claim 1 wherein the engineered RNase 1 retains ribonucleolytic activity, exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2, and has a lower binding affinity for hRI than SEQ ID NO:2.