Protein splicing and auto-cleavage of bacterial intein-like domains lacking a C'-flanking nucleophilic residue.
Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains. The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily. BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity. Here we experimentally study the auto-processing activity of four BIL domains. An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities. The splicing is notable, because this domain has a native Ala C'-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing. B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N' or C' ends. We propose an alternative protein-splicing mechanism for the A-type BIL domains. After an initial N-S acyl shift, creating a thioester bond at the N' end of the domain, the C' end of the domain is cleaved by Asn cyclization. The resulting amino end of the C'-flank attacks the thioester bond next at the N' end of the domain. This aminolysis step splices the two flanks of the domain. The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism. Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing. Yet the predominant reactions diverged in each HINT type according to their specific biological roles. We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins.
Dassa
Bareket
B
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 76100.
Haviv
Haim
H
Amitai
Gil
G
Pietrokovski
Shmuel
S
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry; D015153:Blotting, Western; D002474:Cell-Free System; D002918:Chymotrypsin / Q000494:pharmacology; D003013:Clostridium / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D013058:Mass Spectrometry; D008956:Models, Chemical; D005810:Multigene Family; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014158:Transcription, Genetic; D014357:Trypsin / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; D004247:DNA; D002918:Chymotrypsin; D014357:Trypsin