Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus.
Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases.
Wilde
Christian
C
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.
Just
Ingo
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Aktories
Klaus
K
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001905:Botulinum Toxins; D016297:Mutagenesis, Site-Directed; D009244:NAD+ Nucleosidase / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D009244:NAD+ Nucleosidase; D001905:Botulinum Toxins; D020742:rhoA GTP-Binding Protein