Biochemical and structural analysis of unusual docking domains in non-ribosomal peptide synthetases (NRPS)

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Non-ribosomal peptide synthetases (NRPSs) are the protein templates and catalysts for the biosynthesis of bioactive peptides like daptomycin and cyclosporine, which are of outstanding importance as pharmaceuticals. NRPSs consist of modules, each containing catalytic domains for the incorporation of one amino acid. In bacterial NRPSs the modules are usually spread over more than one polypeptide chain. The intermolecular subunit interactions are crucial for an ordered peptide synthesis and are mediated by various types of small docking domains. This project focuses on the protein-protein-interaction at the interface between epimerization (E) and condensation (C) domains, which is mediated by the so-called COM-domains (communication-mediating). The structure and the specificity-determining amino acids in this region are not or not sufficiently understood, respectively. Since they are at least partially embedded within the large proteins they cannot be studied in isolation. In previous work, we used genetically encoded photo-crosslinkers to derive spatial constraints that supported a helix-hand model of the interaction interface. However, a detailed molecular understanding of the structure is still missing. At the end of the last funding period we succeeded in the first crystallization of a COM domain. Meanwhile, we could solve the crystal structure, which forms the basis for this proposal. In this project, we will focus on the structure-based identification of the specificity-determining residues, their site-directed mutagenesis for the rational re-programming of COM domains and on the possible architectural role of COM domains. Since COM domains are also found in fused form (cis-COM) between internal E and C domains we postulate for the first time a role in the correct spatial interplay of the catalytic domains. We will test if split COM domains (trans-COM) can be generated from cis-COM domains and thereby delineate a conceivable evolutionary development. A detailed structural and biochemical understanding of COM domains will for the first time sufficiently characterize these unusual docking domains. Deciphering the trans-COM domain interactions will be a very important contribution to the combinatorial engineering of NRPSs, in particular because the COM domains hold the key to the E domains, which introduce D-amino acids into the products as unique and important structural elements. We will investigate purified, recombinant proteins as COM-domain model systems and will apply protein crystallography, various biochemical and biophysical methods as well as modern carbene footprinting.