Nonribosomal peptide synthetases (NRPSs): Understanding functional domain interplay and biosynthetic directionality using FRET spectroscopy

• Applicant: Professor Dr. Henning D. Mootz
• Subject Area: Biochemistry ; Biophysics
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 434700456

Nonribosomal peptide synthetases (NRPSs) are large enzymes that act as protein templates for the biosynthesis of many important peptide natural products like daptomycin and cyclosporine. NRPSs show a repetitive modular organization, with each module containing semi-autonomous domains. The domain arrangement dictates the structure of the product, however, the domain interplay remains insufficiently understood. Amino acids are ATP-activated by adenylation (A) domains, bound as acyl thioesters on 4’-phosphopantetheine-peptidyl-carrier proteins (PCP) and linked by condensation (C) domains. Optional domains like the epimerization (E) domain provide structural diversification. A thioesterase domain (TE domain) releases the product. Crystal structures of multi-domain NRPS fragments have provided important static snapshots into the molecular details of domain interactions and possible conformations. However, there is only a very limited understanding of the NRPS conformations adopted in solution and how their dynamics correlate with the stepwise peptide synthesis. Our group has pioneered the first studies towards these questions using FRET spectroscopy (Förster resonance energy transfer), next to complementary approaches. Minimal NRPS FRET sensors were generated by conjugating donor and acceptor fluorophores to interacting pairs of domains. In the last funding period, we have revealed new intermediary conformational states and studied how A and C domains as well as A and E domains compete for the interaction with PCP. Out of the dynamic equilibrium of possible conformations, we have observed different conformational shifts in response to substrates and catalysis steps, that were both expected and unexpected with regard to the synthetic logic. These findings suggest that conformational behavior can contribute in hitherto unpredictable ways to the directionality and (optimized) processivity of NRP biosynthesis. In the new funding period, we will expand our studies to more complex and holistic NRPS systems that include all PCP partner domains. Specifically, we will study conformational changes associated with peptide bond formation in the initiation and further elongation reactions at starter and internal modules, respectively. We will develop the first dimodular NRPS FRET sensors, including novel sensor designs to capture the PCP interaction with its downstream C domain. Furthermore, we will investigate how conformational aspects and potential conformational dead-ends can play out for artificially reprogrammed NRPSs. Finally, we propose a novel labeling strategy of proteins with FRET dyes based on genetic code expansion that can be applied to sensitive proteins such as NRPSs without removal of native cysteines. Together, the proposed work will advance the technology toolbox and will reveal novel key features of conformational dynamics in NRPSs, important in both the native and engineered variants of these complex and fascinating enzymes.

• DFG Programme: Research Grants