Interaction of Bordetella pertussis adenylate cyclase with calmodulin. Identification of two separated calmodulin-binding domains.
The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a photoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:1 complex with CaM or azido-CaM. CaM-bound adenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically active conformation resembling that of the undigested complex. When proteolysis was carried out in the absence of CaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since this fragment cannot be cross-linked with azido-CaM we suggest that the CaM-binding site on the 25-kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001886:Bordetella pertussis / Q000201:enzymology*; D002147:Calmodulin / Q000031:analogs & derivatives / Q000378:metabolism*; D002850:Chromatography, Gel; D004533:Egtazic Acid / Q000494:pharmacology; D008970:Molecular Weight; D010449:Peptide Mapping
D000345:Affinity Labels; D001386:Azides; D002147:Calmodulin; C030955:azidocalmodulin; D004533:Egtazic Acid; D000262:Adenylyl Cyclases