X-ray sequence and crystal structure of luffaculin 1, a novel type 1 ribosome-inactivating protein.

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, The Chinese Academy of Sciences, 155 Yang Qiao Xi Lu, Fuzhou, Fujian, China. houxiaomin@fjirsm.ac.cn <houxiaomin@fjirsm.ac.cn>

BACKGROUND: Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. High resolution X-ray crystallography can also be used to derive protein sequence information, but faces the difficulty in distinguishing the Asp/Asn, Glu/Gln, and Val/Thr pairs. Luffaculin 1 is a new type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Luffa acutangula. Besides rRNA N-glycosidase activity, luffaculin 1 also demonstrates activities including inhibiting tumor cells' proliferation and inducing tumor cells' differentiation. RESULTS: The crystal structure of luffaculin 1 was determined at 1.4 A resolution. Its amino-acid sequence was derived from this high resolution structure using the following criteria: 1) high resolution electron density; 2) comparison of electron density between two molecules that exist in the same crystal; 3) evaluation of the chemical environment of residues to break down the sequence assignment ambiguity in residue pairs Glu/Gln, Asp/Asn, and Val/Thr; 4) comparison with sequences of the homologous proteins. Using the criteria 1 and 2, 66% of the residues can be assigned. By incorporating with criterion 3, 86% of the residues were assigned, suggesting the effectiveness of chemical environment evaluation in breaking down residue ambiguity. In total, 94% of the luffaculin 1 sequence was assigned with high confidence using this improved X-ray sequencing strategy. Two N-acetylglucosamine moieties, linked respectively to the residues Asn77 and Asn84, can be identified in the structure. Residues Tyr70, Tyr110, Glu159 and Arg162 define the active site of luffaculin 1 as an RNA N-glycosidase. CONCLUSION: X-ray sequencing method can be effective to derive sequence information of proteins. The evaluation of the chemical environment of residues is a useful method to break down the assignment ambiguity in Glu/Gln, Asp/Asn, and Val/Thr pairs. The sequence and the crystal structure confirm that luffaculin 1 is a new type 1 RIP.

PMID: 17470286 [PubMed - indexed for MEDLINE]

PMCID: PMC1868734