The Importance of Dynamic Effects on the Enzyme Activity

Abstract

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme.Ribonucleases are widely found in living organisms and have been proposed to function in RNA metabolism and gene expression (1). Several ribonucleases have been isolated from organs of various animals and have been well characterized. Bovine pancreatic ribonuclease (RNase A) 1 is the most studied member of the family (2). It has been used as a model for the study of the fundamental aspects of protein structure and function (2, 3). Several members of the RNase A superfamily exhibit additional biological functions in connection with their intrinsic ribonucleolytic activities (4, 5). In humans, eosinophil-derived neurotoxin and eosinophil cationic protein (6) exert neurotoxicity as well as antiparasitic activity (7). Bovine seminal ribonuclease (BS-RNase) has been found to induce apoptosis of human thyroid carcinoma cell lines (8, 9). Onconase (ONC) from Rana pipiens is cytotoxic (10) and cytostatic to several tumor lines and is currently in phase III clinical trials for the treatment of malignant mesothelioma (8,11,12). ONC shares 30% of identity with the sequence of RNase A, and its three-dimensional structure exhibits a highly conserved ribonuclease-like topology (13), which comprises a characteristic V-shaped ␤-sheet motif surrounded by three ␣-helices. Differences are mainly localized in the loop regions, which are significantly shorter in ONC, and at the C terminus where a disulfide bond, unique to amphibian ribonucleases, tethers the C-terminal residue Cys 104 to Cys 87 located in one strand of the ␤-sheet (13). Another distinct property of ONC is the presence of an N-terminal pyroglutamate (Pyr 1 ), which folds back against the N-terminal helix (13). The overall active site architecture closely resembles that of RNase A and includes His 10 , Lys 31 , and His 97 , which form the catalytic triad corresponding to His 12 , Lys 41 , and His 119 , respectively, of the pancreatic enzyme. Despite this similarity and the activating effe...

Molecular dynamics simulations of the Bcl-2 protein to predict the structure of its unordered flexible loop domain

“…All bonds were constrained using the LINCS algorithm [37], and the simulation was performed under NPT conditions, using the v-rescale coupling algorithm [38] and the Parrinello-Rahman coupling algorithm [39], which stabilized the temperature and pressure (P=1 bar, τ P =0.1 ps; T=300 K, τ T =0.1 ps). A smooth particle mesh Ewald (PME) method [40] was used with a cut-off of 1.4 nm for electrostatic [41] and van der Waals (vdW) [41][42][43] interactions. The electrostatic interactions were calculated with PME using a grid spacing of 0.12 nm.…”

Towards Tricking a Pathogen’s Protease into Fighting Infection: The 3D Structure of a Stable Circularly Permuted Onconase Variant Cleavedby HIV-1 Protease

“…Also, we have solved the three-dimensional structure of ONCFLG. The structural data reported herein for ONCFLG lay the groundwork for the future optimization of zymogens via the redesign of linker length and sequence to better position the hydrogen bond acceptor group to fix the Nξ of Lys57, insertion of additional substitutions such as Met28Leu [40] aimed at increasing the catalytic efficiency or, the design of additional protease cleavage sites within the linker to diminish the steric hindrance of the segments following cleavage. Therefore, the creation of successful zymogens based on ONC would represent a new versatile option to manage the enzymatic activity of this enzyme and target its toxicity to a cell in a specific diseased state.…”

Structural Insight into Anaphase Promoting Complex 3 Structure and Docking with a Natural Inhibitory Compound

“…[48] For the calculation of electrostatics and van der Waals (VDW) interactions with grid spacing of 0.16 nm and 1.4 nm as cut off, the smooth Particle Mesh Ewald method was employed. [49] Finally, the MD simulation was done with a time step of 2.0 fs for 10 ns.…”