Abstract
Onconase (rONC), otherwise known as ranpirnase or P-30 protein, which was initially purified from extracts of Rana pipiens oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo and is in phase III clinical trials for tumor therapy. We have determined the solution NMR structure of a recombinant onconase with Met(-1), Gln1, and Leu23 residues (M-1, Q1, M23L)rONC. The 20 best solution structures had a backbone root mean square deviation of 0.41 +/- 0.09 A with respect to the average structure. The energy-minimized average NMR structure had a backbone root mean square deviation of 0.72 A from the x-ray crystallographic structure of native onconase; however, the orientation of the N-terminal residue in the two structures was very different. Comparison of the 15N HSQC spectrum of (M-1, Q1, M23L)rONC with that of a mutant E1S-rONC, which is identical to the nONC except with the N-terminal pyroglutamyl residue replaced by Ser, showed that N-terminal and residue 23 mutations induced structural changes in regions beyond the mutation sites. Model-free analysis of the backbone amide 15N-T1, 15N-T2, and 15N-1H NOE relaxation data for (M-1, Q1, M23L)rONC and E1S-rONC revealed that the E1S-rONC molecule showed very little flexibility, whereas (M-1, Q1, M23L)rONC exhibited substantial flexibility, which may account for the previously observed reduced stability and increased protease susceptibility. The alpha1 helix and beta-sheets of (M-1, Q1, M23L)rONC displayed bending motions. These data provided strong evidence for the presence of an N-terminal hydrogen bond network in E1S-rONC, but not in (M-1, Q1, M23L)rONC.
Highlights
Onconase, which was initially purified from extracts of Rana pipiens (Northern leopard frog) oocytes and early embryos, exhibits anticancer activity both in vitro and in vivo [1]
A mutant of the recombinant enzyme, (MϪ1, Q1, M23L)rONC, in which a methionine was added to the N terminus, Pyr1 was replaced with Gln, and Met23 was replaced with Leu, shows considerably reduced ribonuclease activity and cytotoxicity; cleavage of the N-terminal Met, followed by cyclization of the new N-terminal Gln residue to the native pyroglutamyl, results in the M23L-rONC mutant, which has an IC50 33.3% of that of native ONC and a catalytic activity 10-fold higher than that of (MϪ1, Q1, M23L)rONC and about 2-fold higher than that of nONC
R. pipiens oocytes; rONC, recombinant onconase; Pyr, pyroglutamyl; M23L-rONC, recombinant onconase with a native N-terminal Pyr1 residue and Met23 mutated to Leu23; (MϪ1, Q1, M23L)rONC, recombinant onconase with MetϪ1, Gln1, and Leu23 residues; E1S-rONC, recombinant onconase with Pyr1 replaced by Ser1; (M-1, E1)rONC, recombinant onconase with MetϪ1, Glu1 residues; RC-RNase, ribonuclease isolated from the oocytes of R. catesbeiana; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; chemical shift indices (CSI), chemical shift index; XNOE, heteronuclear NOE; r.m.s., root mean square; ESI, electrospray ionization; MS, mass spectrometry
Summary
Protein Sample Preparation and Characterization—A synthetic onconase gene coding for (MϪ1, Q1, M23L)rONC, kindly provided by Dr R. Experimental Restraints and Structure Calculation and Analysis—An ensemble of NMR structures of (MϪ1, Q1, M23L)rONC was calculated based on NOE data, backbone dihedral angles, and hydrogen bond constraints. Backbone dihedral angles predicted using TALOS [34] were included in the structure calculation if they were found to be consistent with the secondary structure obtained from the CSI and NOE patterns. These gave rise to a total of 94 and 56 dihedral angle constraints. The NOE cross-peaks assigned by ARIA were interactively reexamined manually using Sparky
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