Solution structure of PCP, a prototype for the peptidyl carrier domains ofmodular peptide synthetases

Background: Nonribosomal peptide synthetases (NRPSs) are large modular enzy mes responsible for the synthesis of a variety of microbial bioactive pepti des, They consist of modules that each recognise and incorporate one specif ic amino acid into the peptide product, A module comprises several domains, which carry out the individual reaction steps. After activation by the ade nylation domain, the amino acid substrate is covalently tethered to a 4'-ph osphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes t he substrate to the reaction centres of the consecutive domains. Results: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techni ques. PCP is a distorted four-helix bundle with an extended loop between th e first two helices, its overall fold resembles the topology of acyl carrie r proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodi n polyketide synthase from Streptomyces coelicolor; however, the surface po larity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same loca tion as in the ACPs and is situated within a stretch of seven flexible resi dues. Conclusions: The structure of PCP reflects its character as a protein domai n. The fold is well defined between residues 8 and 82 and the structural co re of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post- translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.