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

Xiaomin Hou,Edward J Meehan,Liqing Chen,Minghuang Chen,Mingdong Huang,Jieming Xie

doi:10.1186/1472-6807-7-29

Abstract

BackgroundProtein 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.ResultsThe crystal structure of luffaculin 1 was determined at 1.4 Å 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.ConclusionX-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.

Highlights

Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing
We demonstrate that the evaluation of the chemical environments of these pairs can help to break down such ambiguity and 86% of the amino acids were assigned with confidence on the basis of the electron density and the chemical environment evaluation of the residues
We demonstrated that the primary structure of luffaculin 1 can be derived with a high degree of confidence from the high-resolution electron density

Summary

Introduction

Protein sequence can be obtained through Edman degradation, mass spectrometry, or cDNA sequencing. The primary structure of the protein can be obtained through Edman degradation, mass spectrometry or cDNA method. Mass spectrometry has made dramatic advance in the last decade in its technology [2], but still has difficulties to give the full-length sequence and to distinguish the residue pair Ile/Leu. X-ray sequencing method, based on electron density, is another method to determine the protein sequence. X-ray sequencing method, based on electron density, is another method to determine the protein sequence This method has limited usage, it is a useful addition to the sequencing methods in some cases, e.g., where cDNA is not readily available. A major problem of this method is the difficulty to distinguish residue pairs Asp/ Asn, Glu/Gln, and Val/Thr. weak electron density of some residues located at the molecular surface gives rise to uncertainty for the X-ray sequence analysis. We demonstrate that the evaluation of the chemical environments of these pairs can help to break down such ambiguity and 86% (see Table 2) of the amino acids were assigned with confidence on the basis of the electron density and the chemical environment evaluation of the residues

Methods

Results

Conclusion