Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata

A novel type I ribosome-inactivating protein (RIP) designated curcurmosin was isolated from the sarcocarp of Curcurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines Of both human and murine origins, but: low toxicity to normal cells. Plant genomic DNA extracted front the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure Of curcurmosin has been determined at 1.04 A, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven alpha-helices and eight beta-strands, and a smaller C-terminal domain consisting of three of.-helices and two beta-strands. The high resolution structure established a glycosylation pattern of GlcNAc2Man(3)Xyl, Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu 158 and Arg 61 define the active site of cucurmosin as an RNA N-glycosidase. The Structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed. (c) 2008 Elsevier Inc. All rights reserved.