Using secondary structure predictions and site-directed mutagenesis to identify and probe the role of potential active site motifs in the RT6 mono(ADP-ribosyl)transferases.
The RT6 T cell mono(ADP-ribosyl)transferases are expressed as GPI-anchored membrane proteins by mature T lymphocytes. We performed secondary structure prediction analyses of RT6 with a profile based neural network system based on multiple alignments of RT6 with other vertebrate mono(ADP-ribosyl)transferases (mADPRTs). The results reveal a linear order of predicted beta sheets/alpha helix in RT6 that are quite similar to those in the catalytic subunit of the four known crystal structures of mono-ADP-ribosylating bacterial toxins. Recognizable amino acid similarities occur throughout the region of predicted structural homology to the bacterial toxins. Three residues which have been shown to be important for catalysis in bacterial toxins (e.g. R9, S52 and E129 in pertussis toxin) occur in a similar context also in RT6 (R126, S147 and E189). We have mutated these residues in RT6 by site-directed mutagenesis. The RT6 mutants exhibit remarkably similar alterations in enzymatic phenotype as those reported for mutations of the proposed analagous residues in bacterial toxins. These results support the hypothesis that eu- and procaryotic mADPRTs share a common fold and have a common ancestry.
Bredehorst
K
K
Department of Immunology, University Hospital, Hamburg, Germany.
Wursthorn
K
K
Thiele
H G
HG
Haag
F
F
Koch-Nolte
F
F
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte; D001665:Binding Sites; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000945:Antigens, Differentiation, T-Lymphocyte; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D036002:ADP Ribose Transferases; C090163:Art2b protein, rat