Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

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Abstract

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2′-deoxyadenosine triphosphate (2′-dATP) (F). These were determined at 1.67 Å, 1.60 Å, 2.20 Å, 1.70 Å, 2.07 Å and 1.90 Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2′-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ1 =  66°and χ2 = 165° in structures (A) and (B); (ii) χ1 =  95° and χ2 = 70° in structures (C), (D) and (E); and (iii) χ1 =  163° and χ2 = 87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.

Highlights

► The isolated new type 1 ribosome inactivating protein MbRIP-1 is highly toxic. ► Ribose, guanine, ATP and 2′dATP bind to MbRIP-1 at substrate binding site. ► The conformation of active site is unaltered in the complex with ribose. ► The conformation of active site is altered in complexes with guanine and adenine. ► The conformation of active site is further altered when complexed with 2′dATP product.

Introduction

Ribosome inactivating proteins (RIPs) are N-glycosidases (EC. 3.2.2.22) which catalytically hydrolyze N-glycosidic bond between adenine and ribose in highly conserved sarcin/ricin loop of the large subunits of ribosomes [1], [2]. The depurination of this specific adenine results in the prevention of the elongation factor (EF-2) from binding to the large subunits of ribosomes and thus arrests the protein biosynthesis [3]. RIPs are a group of plant toxins and have been classified into three groups according to their structural organizations [4]. Type 1 RIPs, are catalytically active single chain polypeptides with molecular mass ranging from 26 to 31 kDa. Type 2 RIPs with molecular mass ranging from 60 to 65 kDa, consist of two chains, chain A is functionally similar to type 1 RIPs, and is linked through a disulfide bond to lectin-like B chain. Type 3 RIPs are single chain polypeptides containing an extended C-terminal region with an unknown function [5]. The RIPs are known to remove an adenine residue at the 4324th position (according to rat sequence) from the sarcin/ricin loop (SRL) of 28S rRNA of eukaryotic ribosomes. Type 1 RIPs are capable of depurinating both prokaryotic as well as eukaryotic ribosomes [6]. In a contrast, type 2 RIPs have a preference for animal ribosomes [7]. In case of type 3 RIPs, the amino terminal domain resembles with type 1 RIPs. It is linked to an unrelated carboxyl-terminal domain whose function is still unknown [5]. The target site in prokaryotic ribosomes is 2660th adenine residue (according to E. coli sequence) of SRL of 23S rRNA. In addition to SRL, these RIPs also have N-glycosidase activity on other substrates such as, genomic DNA, poly(A)RNA, viral RNA and mRNA [[8], [9]]. It has been suggested that the RIPs have a considerable potential for medicinal applications [10], [11]. They inhibit HIV replication and integration [12], [13] and show specific in vitro and in vivo antitumor activity against breast cancer and melanoma cells [14]. It has been shown to be useful in various clinical conditions [15], [16] while further investigations related to its efficacy against different diseases are required to be pursued. In this regard, it is imperative to examine structures and functions of RIPs from different sources as well as its complexes with various compounds including substrate analogs, substrate products and also structural intermediates. Although structures of type 1 RIPs from several species were known [17], [18], [19], [20], [21], [22], [23], [24], [25], the precise mode of substrate binding and catalytic mechanism was not yet fully understood. Similarly, structures of several type 2 RIPs, whose A chain is structurally and functionally similar to type 1 RIPs, were also known [26], [27], [28], [29] with which a detailed comparison is necessary. We report here crystal structure of a type 1 RIP from Momordica balsamina (MbRIP-1) together with structures of its three complexes, one with ribose sugar, two with free bases guanine and adenine as well as two structures of its complexes with product adenine which was obtained from the crystallization conditions using an RNA substrate adenosine triphosphate (ATP) and a DNA substrate, 2′-deoxyadenosine triphosphate (2′-dATP). These structures have revealed three distinct conformational states of protein representing its unbound state, when bound to free adenine or product adenine from ATP and when bound to product adenine from substrate 2′-dATP. The last structure represents the first RIP structure of a conformational intermediate.

Section snippets

Isolation and purification of a type 1 RIP

The dried seeds of M. balsamina were obtained from local market. The 50 g of seeds was decorticated and pulverized in the presence of liquid nitrogen in a ventilated hood. The 300 ml extraction solution containing 10 mM sodium phosphate buffer (pH 7.5), was added to the ground tissue. The sample was homogenized by Polytron Homogenizer (Cole Parmer Homogenizer, Vernon Hills, USA) for 15 min. The homogenate was stirred for 12 h at 4 °C. In order to remove insoluble particles the homogenate was filtered

Biochemical studies

The purity of samples of MbRIP-1 was established using SDS-PAGE which showed a single band of molecular mass of approximately 30 kDa. The protein was identified as type 1 RIP by determining the sequence of the first 20 amino acid residues, Asp1-Val-Ser-Phe-Arg-Leu-Ser-Gly-Ala-Asp-Pro-Ser-Ser-Tyr-Gly-Met-Phe-Ile-Lys-Asp-Leu20. This sequence showed identities of greater than 90% to other type 1 RIPs such as α-momorcharin [17] and momordin [18]. The complete amino acid sequence (GenBank Accession

Discussion

The active site pocket which is responsible for N-glycosidase activity in type-1 RIPs is situated in a rectangular box formed between two domains. The front wall consists of Tyr70 while back wall is made up of Tyr111. Upper side contains Glu160 and Arg163 while lower part has Glu85 and Gly109. In the unbound state the side chain of Tyr70 is oriented (χ1 =  66° and χ2 = 165°) to an away position from Glu85 and it is toward Arg163. Its OH group is not involved in any intramolecular hydrogen bond. In

Acknowledgements

The authors acknowledge the financial support from the Department of Science and Technology, New Delhi. TPS thanks the Department of Biotechnology, Govt. of India, New Delhi for the award of Distinguished Biotechnology Research Professorship to him.

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