Abstract
Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (KD) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.
Highlights
Ribosome-inactivating proteins (RIPs) comprise more than one hundred N-glycosidases [1] which cleave the N-glycosidic bond of adenine-2660 at 23S rRNA or adenine-4324 at 28S rRNA of ribosome and irreversibly [2,3]
SDAD-labelled Myc-tagged MOD was incubated with rat liver ribosome for interaction and permanent crosslinking was elicited by activation of the diazirine group of SDAD upon UV irradiation
RIPs interact with the sarcin ricin loop (SRL) on the 28S rRNA and remove a specific adenine residue
Summary
Ribosome-inactivating proteins (RIPs) comprise more than one hundred N-glycosidases [1] which cleave the N-glycosidic bond of adenine-2660 at 23S rRNA or adenine-4324 at 28S rRNA of ribosome and irreversibly [2,3]. The corresponding adenine is situated at the a-sarcin ricin loop (SRL) which belongs to the GTPase centre of ribosome and actively involved in the interaction with the elongation factors [4]. Upon depurination of the SRL by RIPs, elongation factors fail to form a stable complex with ribosome and delivery of aminoacyl-tRNA to ribosome and translocation of peptide are prohibited, inevitably causing the cease of protein synthesis and cell death [5,6,7]. Despite sharing highly conserved structure and catalytic centre, the potency and specificity of RIPs on ribosome vary [10,11,12]
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