Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Gorbatyuk, Vitaliy; Tsai, Cheng Kun; Chang, Chi‐Fon; Huang, Tai Huang

doi:10.1074/jbc.m311233200

Cited by 21 publications

(29 citation statements)

References 50 publications

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“…Our present and previous (17,45) results clearly indicate two distinctive features of Onconase with respect to other ribonucleases: very small flexibility and very high thermal stability, both unusual properties for a mesophilic enzyme. The two properties are mainly governed by different structural features.…”

Section: Discussionmentioning

confidence: 70%

“…5A). For a more quantitative estimate we have evaluated the distance between the nitrogen amide atoms of Thr 45 and Phe 120 , two conservative residues placed on the two arms of the ␤-sheet, which contribute with two key hydrogen bonds to the stability of the bound substrate in the active site of RNase A (56, 61) and BS-RNase (62,63). Due to the breathing motion, this distance in the latter proteins depends on several variables, such as crystal packing, crystallization solution, pH, ligands bound to the active site, etc.…”

Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-mentioning

confidence: 99%

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The Importance of Dynamic Effects on the Enzyme Activity

Merlino

Mazzarella

Carannante

et al. 2005

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 70%

Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-mentioning

confidence: 99%

The Importance of Dynamic Effects on the Enzyme Activity

Merlino

Mazzarella

Carannante

et al. 2005

Journal of Biological Chemistry

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“…26,47 This rigidity could hinder facile interaction with the substrate. The structure of the ONC·d(AUGA) complex supports this hypothesis.…”

Section: Rational Design Of Onc Variants With Enhanced Catalytic Actimentioning

confidence: 99%

Structural Basis for Catalysis by Onconase

Lee

Bae

Bingman

et al. 2008

Journal of Molecular Biology

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Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC·nucleic acid complexes: a T89N/E91A ONC·5′-AMP complex at 1.65 Å resolution and a wild-type ONC·d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis was used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH-k cat /K M profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10 2 -fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k cat /K M value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC·d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.

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“…Previous assignments of the 1 H and 15 N resonances of N-ONC had been acquired at 37 °C, pH 3 (14). 3D 15 N-NOESY-HSQC and 3D 15 N-TOCSY-HSQC NMR spectra of a sample of N-ONC were taken at 37 °C, pH 3, prepared by using the procedure outlined in Figure 2A, to confirm previous assignments in 1 H, 15 N – HSQC spectra under these conditions.…”

Section: Methodsmentioning

confidence: 99%

“…By following the continuous change in the positions of the peaks as the temperature was varied, 91 of the 97 previously assigned resonances (14) observed in the 1 H, 15 N – HSQC spectrum were assigned at 16 °C. These assignments were used to identify structure-stabilized resonances along the 3S U -to-3S F pathway.…”

Section: Methodsmentioning

confidence: 99%

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

2011

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In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3SU, to a single structured three-disulfide intermediate species, des-[30–75] or 3SF, at pH 8.0, 25 °C was examined. This transition was first monitored by far-UV CD spectroscopy at pH 8.0, 25 °C, showing that it occurs with formation of secondary structure, presumably due to native interactions. The time-dependence of formation of native-like structure was then followed by NMR spectroscopy after arresting the transition at different times by lowering the pH to 3 and then acquiring 1H,15N – HSQC spectra at pH 3, 16 °C to identify amide hydrogens that become part of native-like structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94 were identified as involved in the earliest detectable native-like structure before complete formation of des-[30–75], and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, the initial, native-like structural elements formed in this transition have been identified, providing details of the initial events in the oxidative folding of onconase.

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Effect of N-terminal and Met23 Mutations on the Structure and Dynamics of Onconase

Cited by 21 publications

References 50 publications

The Importance of Dynamic Effects on the Enzyme Activity

The Importance of Dynamic Effects on the Enzyme Activity

Structural Basis for Catalysis by Onconase

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

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