CcdA chaperones CcdB against irreversible misfolding and aggregation via a cotranslational folding mechanism

Abstract

Cotranslational subunit assembly is thought to be a prominent feature throughout the proteome, but in bacteria, there are only a limited number of experimentally confirmed examples and most involve addition of extraneous tag sequences for experimental convenience. CcdA and CcdB are the antitoxin and toxin components, respectively, of the ccdAB operon. They assemble in a hetero-multimeric complex in vivo. Mutant phenotypes in a saturation mutagenesis library of CcdB were inferred from deep sequencing in two contexts, one when CcdB is expressed alone, and the other when CcdB is present in an operonic context downstream of its cognate antitoxin, CcdA. When expressed in the absence of CcdA, charged and polar mutations in the CcdB core cause the protein to misfold. However, many such deleterious mutations are rescued when expressed along with CcdA in the native operon. CcdA thus acts as a chaperone and relieves the folding defect in CcdBcotranslationally. Assembly efficiency and efficacy decreases when CcdA and CcdB are expressed from separate mRNAs relative to when they are expressed from the same polycistronic mRNA. Gene organisation in operons in bacteria may thus reflect a fundamental cotranslational mechanism that is important for effective assembly of protein complexes and can potentially buffer substantial genetic variation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/618527v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@26e1ceorg.highwire.dtl.DTLVardef@b2a4b1org.highwire.dtl.DTLVardef@5a7c85org.highwire.dtl.DTLVardef@a50512_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefUsing the CcdAB Type II Toxin-Antitoxin system, the authors show that the antitoxin is able to suppress folding defects of the toxin, likely by processes involving cotranslational assembly. HighlightsO_LIDeep mutational scanning is used to assess and compare the foldability of buried-site CcdB mutants in the absence and presence of CcdA. C_LIO_LIFolding defects of CcdB mutants are rescued in the presence of CcdA when expressed in an operonic context. C_LIO_LICcdAB complex formation is enhanced when both the toxin and antitoxin components are synthesized from the same polycistronic mRNA, relative to synthesis from separate mRNAs. C_LIO_LICcdA acts as chaperone both to assist correct folding, and to buffer mutations in CcdB, likely via cotranslational assembly mechanisms. C_LI