Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues.
Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.
Amitai
Gil
G
Department of Molecular Genetics of the Weizmann Institute of Science, Rehovot, Israel 76100.
Dassa
Bareket
B
Pietrokovski
Shmuel
S
D000483:Alleles; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry*; D001419:Bacteria / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D009841:Oligonucleotides; D010455:Peptides; D005973:Glutamine; D001224:Aspartic Acid; D004247:DNA