Clostripain linker deletion variants yield active enzyme in Escherichia coli: a possible function of the linker peptide as intramolecular inhibitor of clostripain automaturation.
The clostripain core protein is composed of the light and heavy chain subunits linked by a nonapeptide into a single polypeptide chain [Mol. Gen. Genet. 240: 140, 1993]. Linker removal is due to autocatalytic processing yielding active heterodimeric enzyme. We have expressed mutationally altered core protein variants in the heterologous host Escherichia coli to gain further insight into the process of clostripain automaturation. In a mutationally created Cys231 --> Ser variant, heterodimer formation was largely impaired, providing molecular evidence that the capacity for automaturation is attributed to the active site cysteine, Cys231, of the native enzyme. Artificially generated deletions of the linker peptide did not prevent the formation of active enzyme. One variant gave rise to a single-chain molecule devoid of the authentic processing sites while retaining enzymatic activity. Experiments performed with linker substitution variants suggested that the efficacy of automaturation depends on a proper configuration of the linker region. According to computerized predictions, the formation of a turn-structured protein loop or hinge with hydrophilic characteristics in the linker region is probably a prerequisite for the interaction of the active site cysteine with the processing sites, Arg181 and Arg190. We propose that the clostripain linker nonapeptide serves as an important transient intramolecular inhibitor in the cellular self-defense program evolved by the natural host Clostridium histolyticum.
Witte
V
V
Weissheimer Research Laboratory, Schaarstr. 1, D-56626 Andernach, Germany.
Wolf
N
N
Dargatz
H
H
D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics* / Q000276:immunology*; D003545:Cysteine / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics*; D010957:Plasmids; D017384:Sequence Deletion
D010455:Peptides; D003546:Cysteine Endopeptidases; C019913:clostripain; D003545:Cysteine