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FRET monitoring of a nonribosomal peptide synthetase

Nature Chemical Biology volume 13, pages 1009–1015 (2017)Cite this article

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Abstract

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine–thioester and phenylalanine–AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

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Figure 1: Design of an intramolecular FRET sensor of an A-PCP di-domain.
Figure 2: The A-PCP FRET sensor undergoes ligand-dependent conformational changes.
Figure 3: Correlation of FRET ratio with catalysis and ligand binding.
Figure 4: A-PCP FRET sensors with selectively destabilized A or T conformations.
Figure 5: Model of the catalytic A-PCP reaction cycle and its coupling to conformational states.

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Acknowledgements

We are grateful to C.-B. Li and S. Kawai for fruitful discussions. We thank J. Diecker for technical support with radioactive assays and FRET experiments and W. Dörner for support with MS measurements. This work was funded by the Human Frontier Science Program (RGP0031/2010 to H.D.M., H.Y. and T.K.) and the National Science Foundation (graduate research fellowship DGE-0646086 to T.E.M.).

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Authors and Affiliations

  1. Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Muenster, Münster, Germany

    Jonas Alfermann, Florian Mayerthaler, Eva Dehling, Gerrit Volkmann & Henning D Mootz

  2. Department of Chemistry, Princeton University, Princeton, New Jersey, USA

    Xun Sun, Thomas E Morrell & Haw Yang

  3. Molecule and Life Nonlinear Sciences Laboratory, Research Center of Mathematics for Social Creativity, Research Institute for Electronic Science (RIES), Hokkaido University, Sapporo, Japan

    Tamiki Komatsuzaki

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  1. Jonas Alfermann
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Contributions

J.A., X.S., T.K., H.Y. and H.D.M. conceived the study. J.A., X.S., F.M., H.Y. and H.D.M. planned the experiments. J.A. developed the FRET sensor and carried out FRET, gel-shift and enzyme assays. X.S. performed MS experiments and photophysical controls of the FRET dye pair. F.M. performed FRET measurements with the desulfo sensor and the AF546 control experiments. E.D. and G.V. established and carried out enzyme assays. T.E.M. performed the computational modeling. J.A., X.S., F.M., H.Y. and H.D.M. interpreted the data. J.A., X.S. and H.D.M. wrote the manuscript.

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Correspondence to Henning D Mootz.

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Alfermann, J., Sun, X., Mayerthaler, F. et al. FRET monitoring of a nonribosomal peptide synthetase. Nat Chem Biol 13, 1009–1015 (2017). https://doi.org/10.1038/nchembio.2435

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