Structure-function study of maize ribosome-inactivating protein: implications for the internal inactivation region and the sole glutamate in the active site

Abstract

Maize ribosome-inactivating protein is classified as a class III or an atypical RNA N-glycosidase. It is synthesized as an inactive precursor with a 25-amino acid internal inactivation region, which is removed in the active form. As the first structural example of this class of proteins, crystals of the precursor and the active form were diffracted to 2.4 and 2.5 Å, respectively. The two proteins are similar, with main chain root mean square deviation (RMSD) of 0.519. In the precursor, the inactivation region is found on the protein surface and consists of a flexible loop followed by a long α-helix. This region diminished both the interaction with ribosome and cytotoxicity, but not cellular uptake. Like bacterial ribosome-inactivating proteins, maize ribosome-inactivating protein does not have a back-up glutamate in the active site, which helps the protein to retain some activity if the catalytic glutamate is mutated. The structure reveals that the active site is too small to accommodate two glutamate residues. Our structure suggests that maize ribosome-inactivating protein may represent an intermediate product in the evolution of ribosome-inactivating proteins.

Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica)

“…Each chain has a molecular weight of $30 kDa and is linked through a disulfide bond (Girbes et al, 2004). Type III RIPs consist of a single enzymatically active polypeptide, but, unlike type I RIPs, are synthesized as zymogens (Mak et al, 2007;Yang et al, 2010).…”

Recent Progress in Medicinal Investigations on Trichosanthin and other Ribosome Inactivating Proteins from the Plant Genus Trichosanthes

“…Type I RIPs consist of a single polypeptide with a molecular weight around 26 to 35 kDa and an alkaline isoelectric point at pH around 8 to 10; Type II RIPs comprise two chains: an A chain with RNA N-glycosidase activity, as well as a cell-binding B chain which binds to the surface of target cells, leading to endocytosis and delivery of the A chain, and resulting in the exhibition of its RNA cleaving activity. Currently, only a few type III RIPs have been reported [13][14][15]. RIPs not only play a role in plant defense but also have potential therapeutic value in humans [16,17].…”

Solution Structure of an Active Mutant of Maize Ribosome-Inactivating Protein (MOD) and Its Interaction with the Ribosomal Stalk Protein P2

“…23 MOD is an active mutant of maize RIP with the central internal inactivation region of Pro-RIP replaced by a 2-residue short linker, and MOD was reported to be about 650 times more active than Pro-RIP by using in vitro ribosomeinactivating activity assay. 24 It is interesting to note that the sequence of the excised 25-residue central region is not homologous to any other published RIP sequence. This process represents a novel mechanism of enzyme activation in plants and resembles processing of certain human hormones, such as insulin.…”

“…Recently, the crystal structures of ΔN5-MOD and ΔN5-Pro-RIP variants have been reported, 24 where ΔN5 represents the fact that the first five residues in the N terminus have been deleted. Although ΔN5-MOD has a structure very similar to other RIPs, it does show some significant structural differences, including missing α-helix B and β-strand in the N-terminal domain and the replacement of β-strands 7 and 8 in the C-terminal The ribbon representations of the lowest-energy model of MOD.…”

“…Since β-strands 7 and 8 are the reported P2 binding site on TCS 29 and the internal inactivation region in maize Pro-RIP is located at the N-terminal domain, it is speculated that the interaction of MOD with ribosome may be different from that of other RIPs. 24 In order to rectify these structural changes and to characterize their effects against P2 binding, it has been our endeavor to determine the 3D structure of this unusual type III RIP, MOD, in solution and to investigate its interaction with the P2 protein by NMR spectroscopy. Solution structure determination of MOD Recombinant 15 N/ 13 C-labeled MOD was overexpressed in E. coli and the structure was determined using heteronuclear multidimensional NMR spectroscopy.…”