Amino Acid Sequence, S-S Bridge Arrangement and Distribution in Plant Tissues of Thionins from Viscum album

Abstract

The complete primary structure of a cytotoxic 5 kDa polypeptide, viscotoxin A1, isolated from Viscum album L., has been determined by combining classical Edman degradation methodology with advanced mass spectrometric procedures. The same integrated approach allowed correction of the sequence of viscotoxin A2 and definition of the pattern of the disulfide bridges. The arrangement of the cysteine pairing was determined as Cys3-Cys40, Cys4-Cys32 and Cys16-Cys26. The primary structure of viscotoxin A1 shares a high degree of similarity with the known viscotoxins and more generally with the plant alpha- and beta-thionins. The pattern of S-S bridges determined for viscotoxin A2 and A1 is similar to that inferred by X-ray and NMR analysis in crambin and related to that present in alpha-purothionin and beta-hordothionin, thus indicating a highly conserved organization of the S-S pairings within the entire family. This arrangement of S-S bridges describes a peculiar structural motif, indicated as 'concentric motif', which is suggested to stabilize a common structure occurring in various small proteins able to interact with cell membranes. The distribution of the new variant toxin in different mistletoe subspecies was investigated. Viscotoxin A1 is abundant in the seeds of the three European subspecies of V. album whereas it represents a minor component in the shoots.

Plant thionins – the structural perspective

“…Several experiments suggest an important role in defense against pathogenic invaders [37][38][39]. Thionin synthesis in response to bacterial invasion [40][41][42], its accumulation in vulnerable tissues [15,[43][44][45], its toxicity to different organisms and cell lines [37,[46][47][48][49], and the improved resistance observed when expressed transgenetically [50][51][52], all strongly support this function. However, their high concentration in the endosperm (in some species up to 10% of the seed mass) and high cysteine content suggest that they also serve as storage proteins.…”

Automated 2D NOESY Assignment and Structure Calculation of Crambin(S22/I25) with the Self-Correcting Distance Geometry Based NOAH/DIAMOD Programs

“…Crambin, isolated from the seeds of Crambe abysinica, is a 46-residue protein with unusually high solubility in ethanol and organic solvents. This unusual solubility of crambin, as well as its homology to membrane active plant toxins (such as purothionins (23)), has excited much interest in the structure/ function relationship of crambin. The recent expression of crambin as a fusion protein in Escherichia coli (24) means that many mutants of crambin should soon be available for structural analysis, suggesting an immediate use for a good automated method for data assignment and interpretation.…”

Clustering of non-polar contacts in proteins.

“…When the classification of residues into non-polar clusters is compared to a multiple alignment of the protein sequences of crambin, viscotoxins and thionins (Orrù et al, 1997), it is seen that most conserved residues are found in the two main clusters identified in this analysis.…”

“…Crambin is a hydrophobic 46 residue protein (Teeter et al, 1981) with sequence similarity to thionins (Orrù et al, 1997), a family of membrane-active toxins from plants. Crambin is found in the seeds of Crambe abyssinica, but the function of crambin in these seeds is still unknown.…”