Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity.
Bryodin 1 (BD1) is a type I ribosome-inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBD1) has been produced and crystallized. To gain insight into the relationship of rBD1 structure and function, we investigated the role of sequences in a region (residues 128-156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site. Progressive deletions representing alpha-helical tums within these residues were generated; mutant rBD1 proteins were expressed in Escherichia coli and demonstrated increasing losses of enzymatic activity. Point mutations were also generated within this region to replace Y140, Y141, and Y142 with either alanine or lysine. Mutants at position 140 or 142 retained full enzymatic activity, whereas A141 and K141 mutants were >19-fold less potent. In cytotoxicity assays, the rBD1 point mutants at Y141 were >80-fold less potent than either rBD1 or mutants at residues 140 or 142. However, when introduced into the anti-CD40 single-chain immunotoxin rBD1-G28-5 sFv, the A140 and A141 point mutations led to decreased cytotoxicity toward CD40 positive cell lines. These data indicate that Y141 plays an important role in the enzymatic activity of BD1 and that Y140, although not essential for catalytic activity, is required for full BD1 function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.
Fryxell
D K
DK
Molecular Immunology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
Gawlak
S L
SL
Dodge
R W
RW
Siegall
C B
CB
D000595:Amino Acid Sequence; D001665:Binding Sites; D019013:CD40 Antigens / Q000276:immunology; D002384:Catalysis; D002470:Cell Survival / Q000187:drug effects*; D002474:Cell-Free System; D002942:Circular Dichroism; D000922:Immunotoxins / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D017354:Point Mutation; D011487:Protein Conformation; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological
D019013:CD40 Antigens; D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; C051829:bryodin