Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics1. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites2,3. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space3,4. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase5,6. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.
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We thank T. Mintya, D. Avizonis, M.-C. Tang and A. Furtos for experimental support, R. Zamboni, J. Collucci and K. Guerard for small molecule synthesis assistance; J. Jiang, D. Alonzo and D. Rodinov for experimental advice and assistance; S. Labuik and P. Grochulski (Canadian Light Source) for diffraction data collection; R. Gillian (CHESS SAXS beamline G1), M. Pillon and A. Guarne for SAXS help: K. Bloudoff, M. Tarry, A. Haque and A. Beghuis for helpful discussions; and J. Pelletier, N. Rogerson and A. Nahvi for critical reading of the manuscript. This work was supported by CIHR grant 106615, a HFSP CDA and a Canada Research Chair in Macromolecular Machines to T.M.S. J.M.R. is supported by an NSERC Alexander Graham Bell studentship, and M.N.A. by a studentship from the CIHR Training Grant in Chemical Biology.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Synthetic cycles in canonical initiation, canonical elongation and LgrA initiation modules.
Schematic diagrams comparing the synthetic cycle in canonical initiation and elongation modules with that in the LgrA initiation module.
a–d, 2Fo − Fc density maps for protein in P41212 (a), R3:H (b), P21 (c) and P322 (d) crystal forms contoured at 1σ. e, f, Unbiased Fo–Fc density maps for the PPE–NH–Val arm in the P21 (thiolation state) contoured at 3.3σ (e), and a P41212 crystal soaked with N5–f THF, AMPcPP and valine contoured at 2.5σ (f).
a–f, Models of F–A (Asub disordered) (a), F–A–PCP (PCP disordered) (b–d) and F–A–PCP from the four independent crystals structures determined (e, f). The crystal with space group P322 diffracted anisotropically to ~3.8 Å resolution, but the other higher resolution structures enabled the building of high quality models shown in d and f.
Extended Data Figure 4 Comparison between the LgrA initiation module and the SrfAC termination module.
a, b, The LgrA initiation module in the formylation state (a) and the termination module of surfactin synthase subunit 3 (SrfAC)22 (b) in the state where aminoacyl-PCP would be positioned to act as an acceptor substrate in the condensation reaction (PPE arm not present). The F and C domains are each positioned directly N-terminal of their A domains and bury similar amounts of A domain surface area (829 Å2 and 903 Å2; contributing residues shown in spheres), each forming ‘stable platforms’22. Both modules use very large movements of their PCP and Asub domains to bring the aminoacyl-PCP of the module to distant active sites to act as the acceptor substrate in an amide bond forming reaction. c, However, the F–A and C–A interfaces are distinct, and, if the A domains are superimposed, the F and C domains are only partially overlapping. This places their active sites in dissimilar locations, necessitating that Asub and PCP assume different positions to deliver their substrate. The PCP domain in the formylation state completely overlaps with the position of the C domain.
a, The crystal structure in the formylation state is shown superposed on the averaged filtered ab initio small-angle X-ray scattering model generated with DAMAVER37, with a NSD value of 0.819 ± 0.052. b, The calculated scattering curve for the DAMAVER is overlaid with the experimental scattering with χ2 = 3.010, where I represents scattering intensity and q is equivalent to 4πsin(θ)/λ. c, To understand the flexibility of F–A–PCP better, EOM39 was performed and generated five different ensembles. The ensemble resembling the formylation state structure represented over 60% of the optimized models generated, while the remaining <40% resembled the thiolation state structure. d, The calculated scattering of the EOM model has a χ2 = 1.028, which demonstrates that F–A–PCP has flexibility. The data are consistent with extreme flexibility for Asub and PCP domains, and limited flexibility in F–Acore. e, All independent molecules from the crystal structures were overlaid to further illustrate the flexibility of the system. f, CRYSOL38 was used to generate predicted scattering curves for the formylation state and thiolation state crystal structures with χ2 = 2.12 and χ2 = 5.54, respectively. Source data
This neighbour-joining tree of the LgrA F domain and homologues was made using PHYLIP (http://evolution.genetics.washington.edu) based on an initial Clustal Omega48 alignment of the closest 220 homologues of the LgrA F domain (Blast50 BLAST E-value <1 × 1014). The most similar formyltransferases to the F domain share ~45% identity, and all of these 220 formyltransferases have only inferred function. The tree was drawn using the program FigTree (http://tree.bio.ed.ac.uk). The sequences are named with their GenInfo Identifier (GI) numbers. Colouring: red, Brevibacilli; green, other Firmicutes; black, other bacteria; blue, Archaea. The clade of the LgrA F domain is highlighted in grey. Only nodes with bootstraps of >50% are shown. Several horizontal transfer events are evident where Firmicute and non-Firmicute proteins cluster together with high bootstrap values (for example, >70%). The several horizontal transfer events of formyltransferase domains between Firmicutes and other bacterial groups suggest the LgrA F domain likely originated from horizontal transfer.
This neighbour-joining tree of LgrA A–PCP didomains and homologues was made for the 500 closest homologues (BLAST E-value <1 × 1014). The sequences are named with their GI accession codes. Colouring: red, Brevibacilli; green, other Firmicutes; black, other bacteria. The significant clades of the LgrA A–PCP domains are highlighted in grey. Only nodes with bootstraps of >50% are shown. Three functionally characterized homologues of LgrA that are shown to be directly related are labelled. The A–PCP portion of the initiation module is quite divergent, but the second module of LgrA clearly shares a common origin with functionally characterized NRPSs in Bacilli and other Firmicutes.
Extended Data Figure 8 Conservation and variation of residues involved in the interaction interfaces.
a, b, Sequence logos made using the WebLogo server (http://weblogo.berkeley.edu)52 show conservation and variation as found in multiple sequence alignments of F domain residues that interact with the A domain (a) and A domain residues that interact with the F domain (b). Below each logo are the corresponding residues in the LgrA proteins from the five Brevibacillus species, with the crystallized LgrA on the first line. FT, formyltransferase. c, d, Sequence logos indicate the conservation and variation in F domain residues involved in binding and interaction with PCP–PPE–Val across the closest 240 homologues of LgrA (c) and all of the functionally or structurally characterized formyltransferase proteins (d) (reduced for redundancy so that no two sequences have >50% sequence identity). e, Consensus sequences for the five Brevibacillus LgrA homologues and for the formyltransferases of known structure for each of three formyltransferase types. Catalytic residues are His73, Asn71 and Asp108.
a, b, The Asub (a) and PCP2,25,54 (b) domains must maximize the use of their limited surfaces to interact with their many binding partners. Shown are the surfaces observed in this study, and many excellent previous studies have also documented interaction surfaces biochemically or structurally. This includes, for example, the equivalent of PCP domain residues Met249, Phe264 and Ala268, which are required for interaction with the C domain in the acceptor site55 and form hydrophobic interactions with the C domain22 in a very similar manner and using an overlapping surface, as the PCP domain does to interact with the F domain. Furthermore, partially overlapping surfaces in PCP domains have been proposed to interact with their (acyl-)PPE arm to protect thioester intermediates56 or to promote binding to the appropriate partner domain57. These interactions might occur during PCP domain transit, but they would have to be broken before productive binding to partner domains. Several of these PPE interactions are incompatible with the productive domain–domain interactions57, and in catalytic configurations seen here and previously, the PPE arms extend into the partner domain and make little contact with the PCP domain.
This file contains the legend for Supplementary Videos 1 and 2 with additional references. (PDF 148 kb)
The structures of the initiation module of LgrA interpolated to visualize its entire synthetic cycle. The A domain is open for substrate binding (see also A domain structures2,20), and closes to catalyze valine adenylation formation (see also A domain structures2,18). Next, the Asub domain rotates 140° to catalyze transfer of the valine onto the thiol of the PPE arm of the PCP domain (see also A-PCP didomain structures19,21). Then the PCP domain transports its valine the 50 Å to the F domain to accept a formyl group. The PCP next moves the formyl-valine to the downstream elongation module, where it is passed off in that module’s condensation reaction (not shown), which liberates the PCP to participate in the next round of reactions. The experimentally observed structures are indicated by crystallographic statistics (disordered or absent portions for each structure coloured grey). For this animation, details of the adenylate in the A domain and the valine in formylation active site were based on co-complexes of isolated A domains58-62, A-PCP domains21,63,64 and FT proteins5,13,15,24. The extensive body of NRPS structural biology of protein constructs that contain A and/or PCP domains was also checked17,21,22,25,58-73 (see separate Supplementary Information file for additional references). (MOV 27353 kb)
The structures of the initiation module of LgrA interpolated to visualize its entire synthetic cycle. The A domain is open for substrate binding (see also A domain structures2,20), and closes to catalyze valine adenylation formation (see also A domain structures2,18). Next, the Asub domain rotates 140° to catalyze transfer of the valine onto the thiol of the PPE arm of the PCP domain (see also A-PCP didomain structures19,21). Then the PCP domain transports its valine the 50 Å to the F domain to accept a formyl group. The PCP next moves the formyl-valine to the downstream elongation module, where it is passed off in that module’s condensation reaction (not shown), which liberates the PCP to participate in the next round of reactions. The experimentally observed structures are indicated by crystallographic statistics (disordered or absent portions for each structure coloured grey). For this animation, details of the adenylate in the A domain and the valine in formylation active site were based on co-complexes of isolated A domains58-62, A-PCP domains21,63,64 and FT proteins5,13,15,24. The extensive body of NRPS structural biology of protein constructs that contain A and/or PCP domains was also checked17,21,22,25,58-73. (see separate Supplementary Information file for additional references). (MOV 29669 kb)
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Reimer, J., Aloise, M., Harrison, P. et al. Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase. Nature 529, 239–242 (2016). https://doi.org/10.1038/nature16503
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