Structures of two distinct conformations of holo-non-ribosomal peptide synthetases

Abstract

Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs)1,2,3,4. During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion5. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases.

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Figure 1: Ribbon diagrams of complete NRPS modules.
Figure 2: NRPS domain structures.
Figure 3: Conformational dynamics in NRPS modules.
Figure 4: Dynamics of the NRPS cycle.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank (PDB) under accession numbers 4ZXH (holo-AB3403), 4ZXI (holo-AB3403 bound to AMP and glycine), and 4ZXJ (holo-EntF bound to Ser-AVS).

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Acknowledgements

We thank R. Sanishvili for assistance with data collection. This work was funded in part by National Institutes of Health GM-068440 (to A.M.G.) and GM-115601 (to G.S.), and Award W81XWH-11-2-0218 from the Telemedicine and Advanced Technology Research Center of the US Army Medical Research and Materiel Command (A.M.G.). Data were collected at the GM/CA beamline of the Advanced Photon Source, which is funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006) under Department of Energy contract number DE-AC02-06CH11357 to A.P.S. A Stafford Fellowship (to B.R.M.) and support from the Hauptman-Woodward Institute is acknowledged.

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C.L.A. characterized activity of and initially crystallized AB3403. J.A.S. initially crystallized EntF. E.J.D. and B.R.M. optimized crystal, and solved and refined the models of AB3403 and EntF, respectively. C.S. and C.C.A. designed and synthesized the mechanism-based inhibitor. J.T.T. and G.S. performed and analysed the single-particle electron microscopy. A.M.G., E.J.D., B.R.M., G.S., J.T.T., C.C.A., and C.S. analysed the results and wrote the manuscript. All authors saw and approved the manuscript.

Corresponding author

Correspondence to Andrew M. Gulick.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Structure-based alignment of EntF, AB3403, and SrfA-C.

Condensation, adenylation, PCP, and thioesterase domains are represented with bars in grey, pink, green–cyan, and blue. Conserved motifs and catalytically important residues are highlighted with the same colours, including the HHxxxD motif of the condensation domains, the aspartic acid hinge that separates the N- and C-terminal subdomains of the adenylation domain, the GGHS motif that is the site of pantetheinylation in the PCP, and the catalytic nucleophile of the thioesterase domain. The SrfA-C, AB3403, and EntF proteins share approximately 26% sequence identity. The adenylation and PCP domains are more well-conserved, sharing ~35% identity, whereas the condensation (21%) and thioesterase (25%) domains are less well conserved. Domain boundaries are described in the table below.

Extended Data Figure 2 Substrate specificity of full-length AB3403.

Amino-acid specificity of AB3403 was recorded for all 20 proteinogenic amino acids, as well as 4-chlorobenzoate (4CB) and 4-hydroxybenzoate (4HB). Average values and standard deviations are shown for three replicates with each substrate; results were recorded as micromoles of radiolabelled ATP incorporated per minute per milligram of enzyme. Apparent kinetic constants are also shown for ATP and glycine calculated from duplicate measurements for four to six substrate concentrations.

Extended Data Figure 3 Stereo representations of electron density figures shown in Fig. 2.

To better visualize the active sites and electron density quality, stereo figures are included in the extended data. In all panels, density is shown with coefficients of the form (Fo − Fc) calculated before inclusion of ligands and contoured at 3σ. a, Stereo representation of electron density of AB3403 condensation domain shows the phosphopantethine on Ser1006 approaching His145 within the condensation domain pocket. Inhibitor carbon atoms in green, carbons of residues within 5 Å of inhibitor in grey, nitrogen in blue, oxygen in red, sulphur in yellow, and water in light blue. b, Electron density of the nucleotide binding pocket of AB3403 bound to glycine and AMP. Stereo representation of electron density shows the AMP, glycine, and Mg+ present in the active site of the adenylation domain. Ligand carbon atoms are in green, carbons of residues within 5 Å of inhibitor in grey, nitrogen in blue, oxygen in red, phosphorus in orange, and the Mg+ cofactor in purple. c, Stereo representation of the electron density shows the phosphopantethine on Ser1006 covalently attached to the Ser-AVS inhibitor in the active site of the adenylation domain. Inhibitor carbon atoms in green, carbons of residues within 4 Å of inhibitor in grey, nitrogen in blue, oxygen in red, phosphorus in orange, sulphur in yellow, and water in light blue.

Extended Data Figure 4 Comparison of AB3403 and SrfA-C PCP-condensation domain interaction.

Stereo representation illustrating different orientations of the PCP domains of SrfA-C and AB3403 relative to the condensation domains with which they interact. AB3403 is shown with a white condensation domain and a green-cyan PCP. SrfA-C is shown with a yellow condensation domain and a pale blue PCP. The pantetheine of AB3403 is shown bound to Ser1006. The position of Ser1003, mutated to an alanine residue in SrfA-C, is also highlighted.

Extended Data Figure 5 Comparison of AB3403 thioesterase domain to the functional PCP–thioesterase interaction.

Stereo representation of the thioesterase (blue) domain of AB3403 interacts with the back face of the PCP domain in AB3403. The functional interaction between the EntF thioesterase domain and its holo-PCP, trapped crystallographically, illustrates that the same face of the thioesterase domain interacts functionally (PDB 3TEJ). A 28-residue insertion of AB3403 is coloured yellow.

Extended Data Figure 6 Synthesis of Ser-AVS.

The Ser-AVS probe was synthesized following similar protocols described elsewhere41,46. Garner’s aldehyde 1 was coupled with 2 using LiHMDS to exclusively furnish the (E)-vinylsulfonamide 3. Mitsunobu coupling of 3 with bis-Boc adenosine 4 afforded 5, which was globally deprotected using 80% aqueous trifluoroacetic acid to yield Ser-AVS.

Extended Data Figure 7 Electrophoretic mobility of EntF.

a, Native gel electrophoresis. Lane 1: EntF. Lane2: EntF incubated with fourfold molar excess of Ser-AVS inhibitor. Lane 3: EntF Crystals. Lane 4: novex NativeMark labelled in kilodaltons. b, Denaturing gel electrophoresis using loading buffer with SDS and β-mercaptoethanol. Gel lane 1: EntF. Lane 2: EntF incubated four times with Ser-AVS inhibitor. Lane 3: Life Technologies Mark12 labelled in kilodaltons. The native gel shows the inhibited EntF in a more compact conformation compared with EntF without the inhibitor.

Extended Data Figure 8 Negative-stain electron microscopy analysis of EntF.

a, Raw electron microscopy image of negative-stained EntF. b, Class averages of EntF particles.

Extended Data Table 1 Diffraction data statistics and refinement statistics for AB3403
Extended Data Table 2 Diffraction data statistics and refinement statistics for EntF

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Drake, E., Miller, B., Shi, C. et al. Structures of two distinct conformations of holo-non-ribosomal peptide synthetases. Nature 529, 235–238 (2016). https://doi.org/10.1038/nature16163

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