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Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs (NDDs) as well as of an NDD-CDD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.
RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts.