Structural Basis for Catalysis by Onconase

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Abstract

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 were 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 versus kcat/KM 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 102-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 kcat/KM 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.

Introduction

The viability of organisms relies on the ability of proteins to recognize nucleic acids. In contrast, the ability of an enzyme to both recognize a nucleic acid and catalyze its cleavage can have deleterious consequences. For example, ribonucleases can be cytotoxic because cleaving RNA renders indecipherable its encoded information.1, 2

Onconase® (ONC; Figure 1(a)) is a ribonuclease found in the eggs and early embryos of the frog Rana pipiens. ONC is a homolog of bovine pancreatic ribonuclease (RNase A),3 and the two proteins share 30% amino acid sequence identity and a similar three-dimensional structure.4 ONC is in confirmatory phase IIIb clinical trials for the treatment of malignant mesothelioma,5, 6 and has been granted both orphan drug and fast track status by the US Food and Drug Administration. ONC also inhibits human immunodeficiency virus type 1 replication.7

ONC is a unique ribonuclease. The protein exhibits remarkable conformational stability (Tm = 90 °C).8 Four disulfide bonds and the absence of a cis-prolyl peptide bond contribute to this attribute.8, 9, 10, 11 ONC evades the cellular ribonuclease inhibitor protein (RI),12 to which other ribonucleases bind with femtomolar affinity.13, 14, 15, 16, 17, 18, 19 The exceptional conformational stability and the RI-evading ability contribute to its cytotoxic activity.8, 17, 20

ONC is a poor catalyst. The ribonucleolytic activity of ONC is three to five orders of magnitude lower than that of RNase A due, in large part, to low affinity for its substrate.21 Nonetheless, the catalytic activity of ONC is necessary for cytotoxicity.22

Homologs of RNase A bind a pyrimidine residue on the 5′ side of the scissile phosphodiester bond in a small, conserved nucleobase-binding site.23, 24, 25, 26 ONC displays a distinct preference for a guanosine nucleoside on the 3′ side of the scissile phosphodiester bond.21 This guanine preference is found for other frog ribonucleases,27 but not in mammalian homologs.28 The basis for this guanine preference in ONC is unknown. tRNA appears to be the major cellular substrate for ONC.29 A recent study revealed an unconventional cleavage sequence for ONC: the guanosine–guanosine phosphodiester bond in the variable loop or the D-arm in tRNA.30

Here, we report the first crystal structures of ONC–nucleic acid complexes. We use this structural information to address key issues in ONC catalysis. First, we determine the molecular basis for the nucleobase specificity of ONC through a systematic site-directed mutagenesis study. Next, we ask whether the low level of catalytic activity of ONC can be enhanced by a rational design approach. Finally, we seek to confirm the cellular target sequence of ONC in vitro using two novel fluorogenic substrates. We anticipate that the development of ONC as a cancer chemotherapeutic will benefit from the incipient understanding of its catalysis.

Section snippets

Structural overview

The crystalline structures of the T89N/E91A ONC–5′-AMP and ONC–d(AUGA) complexes were solved to a resolution of 1.65 Å and 1.90 Å, respectively. Data collection, refinement, and model statistics are summarized in Table 1. The electron density was continuous for main-chain and side-chain atoms. Asymmetric units of the structures contain a single monomer with a chain-fold virtually identical with that of free ONC (Protein Data Bank (PDB) entry 1ONC).4 Both structures exhibit the typical bilobal

Molecular basis for the B2-subsite specificity of ONC

The contribution hydrogen bonds in an enzyme–substrate interface to catalysis has been established.55, 56, 57, 58, 59 In the structure of an ONC–nucleic acid complex, Oε1 and Oε2 of Glu91 form two hydrogen bonds with N(1) and N(2) of the guanine nucleobase (Figure 1(d)). In addition, Thr89 is located proximal to the guanine nucleobase. The distances between Oγ1 and Cγ2 of Thr89, and O(6) of guanine are 3.57 Å and 4.25 Å, respectively. Similar interactions were observed in the RC-RNase·d(ACGA)

Conclusions

We have determined the crystalline structure of two ONC–nucleic acid complexes at a resolution of 1.90 Å and 1.65 Å. Guided by these structures, we have revealed the atomic basis for substrate recognition and turnover by ONC. We have discovered that ONC utilizes Coulombic interactions (especially from Glu91) and a hydrogen bonding network to mediate substrate specificity, and have demonstrated that rational amino acid substitutions can alter this specificity. Finally, we have probed structural

Materials

Human RI (RNasin®) was from Promega (Madison, WI). RNase T1 was from Ambion (Austin, TX). 6-Carboxyfluorescein–dArUdAdA–6-carboxytetramethylrhodamine (6-FAM–dArUdAdA–6-TAMRA), 6-FAM–dArUdGdA–6-TAMRA, 6-FAM–dUrGdGdA–6-TAMRA, and 6-FAM–dArGdGdA–6-TAMRA were from Integrated DNA Technologies (Coralville, IA). Mes, 5′-AMP, and 5′-GMP were from Sigma Chemical (St. Louis, MO). Mes was purified further by anion-exchange chromatography to eliminate contaminating oligo(vinylsulfonic acid), which is a

Acknowledgements

We are grateful to J. B. Binder for help with calculations of the electron density of nucleobases, E. L. Myers and T. J. Rutkoski for contributive discussions, and members of the Center for Eukaryotic Structural Genomics, including L. Meske and A. Hibbard for help with crystallization, and E. Bitto and J. G. McCoy for help with data collection and processing. J.E.L. was supported by a Steenbock Predoctoral Fellowship from the Department of Biochemistry. This work was supported by grant CA73808

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    J.E.L. and E.B. contributed equally to this work.

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