Assignment of functional domains involved in ADP-ribosylation and B-oligomer binding within the carboxyl terminus of the S1 subunit of pertussis toxin.
The roles of the carboxyl terminus of the S1 subunit (composed of 235 amino acids) of pertussis toxin in the ADP-ribosylation of transducin (Gt) and in B-oligomer binding were defined by analysis of two carboxyl-terminal deletion mutants of the recombinant S1 (rS1) subunit: C204, which is composed of amino acids 1 through 204 of S1, and C219, which is composed of amino acids 1 through 219 of S1. C204 was expressed in Escherichia coli as a stable, soluble peptide that had an apparent molecular mass of 23.4 kDa. In a linear velocity assay, the specific activity of C180 was 2% and that of C204 was 80% of the activity displayed by rS1 in catalyzing the ADP-ribosylation of Gt. In addition, C204 possessed catalytic efficiencies (kcat/Km) that were 110% at variable Gt concentrations and 40% at variable NAD concentrations of those reported for rS1. These data showed that the catalytic activity of C204 approached the activity of S1. C204 and C219 were unable to associate with the B oligomer under conditions which promoted association of rS1 with the B oligomer. Consistent with these results, mixtures of C204 or C219 with the B oligomer did not elicit a clustering phenotype in CHO cells, whereas rS1 which had associated with the B oligomer was as cytotoxic as native pertussis toxin. These data indicate that residues between 219 and 235 are important in the association of the S1 subunit with the B oligomer. These data allow the assignment of functional regions to the carboxyl terminus of S1. Residues 195 to 204 are required for optimal ADP-ribosyltransferase activity, residues 205 to 219 link the catalytic region of S1 and a B-oligomer-binding region of S1, and residues 220 to 235 are required for association of S1 with the B oligomer.
Krueger
K M
KM
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbieri
J T
JT
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D037342:Pertussis Toxin; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000737:chemistry* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C083388:pertussis toxin, S1 subunit; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin