10 11 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Macromolecular Crystallography Group Beamlines I02, I03, I04 and I24 Diamond shines light on peptide antibiotic biosynthesis Related publication: Ghilarov D., Stevenson C. E. M.,Travin D.Y., Piskunova J., SerebryakovaM., Maxwell A., Lawson D. M. & Severinov K. Architecture of microcin B17 synthetase: an octameric protein complex converting a ribosomally synthesized peptide into a DNA gyrase poison. Mol. Cell. 73 , 1-14 (2019). DOI: 10.1016/j.molcel.2018.11.032 Publication keywords: Antibiotic; Protein complex; Crystal structure; Escherichia coli; Microcin B17; DNA gyrase; Azole synthetase; Heterocyclase; Dehydrogenase;Topoisomerase N ew antibiotics are urgently needed to counter the ongoing threat of antimicrobial resistance. One such potential antibiotic called microcin B17 (MccB17) has gained attention for its distinct mechanism of action that allows it to remain effective against certain resistant bacteria. It is a peptide-derived antibiotic that targets DNA gyrase to cause double-strand breaks in DNAwithin pathogenic bacteria. MccB17 is produced by a large, multiprotein complex known as microcin B17 synthetase, which is found in E.coli . To fully understand how MccB17 and related antibiotics are made, a series of X-ray Macromolecular Crystallography (MX) datasets from crystals of the synthetase were collected at MX beamlines I02, I03, I04 and I24 at Diamond Light Source. The crystal structures revealed that microcin B17 synthetase was made of eight protein subunits, which can be divided into two halves, each containing distinct active sites. Using these structural data, it was hypothesised that MccB17 flips from one active site to the other as it is modified from a harmless peptide into an antibiotic. AlthoughMccB17has been shown tohaveproperties thatmake it unsuitable for use as anantibiotic inhumans, it is hoped that this increased understanding could inform future therapeutics. The modifications observed in MccB17 could be introduced to other peptides to produce a new generation of novel antibiotics. DNA gyrase is an essential enzyme found in gram-negative bacteria and represents a well validated target for antibiotics 1 . However, the emergence of pathogenic strains that have developed resistance to clinically important compounds that target this enzyme, most notably the fluoroquinolones, has driven the search for new molecules with therapeutic potential. Microcin B17 (MccB17) isapeptide-derived inhibitortargetingDNAgyraseproducedby E.coli to give it a selective advantage over competing enteric bacteria; the inhibition of DNA gyrase by MccB17 leads to the accumulation of double-stranded DNA breaks and cell death 2 . Whilst the details are not fully understood, the mechanism of action of MccB17 is distinct from that used by fluoroquinolones and it is therefore likely to remain effective against fluoroquinolone-resistant mutants of DNA gyrase. Whilst MccB17 itself is unlikely to find clinical use due to unfavourable pharmacokinetics, a detailed understanding of how it is made could inspire the generation of novel therapeutic leads. The biosynthesis of MccB17 is encoded by the plasmid borne mcbABCDEFG cluster, where McbA is a 69-amino acid precursor peptide which is post- translationally modified through the concerted action of McbB, McbC and McbD, which are collectively described as the McbBCD synthetase complex. McbA is a serine- and cysteine-rich peptide, and these residues are transformed into five-membered oxazole and thiazole rings, respectively, via sequential heterocyclisation and oxidation reactions (Fig. 1). Thus, MccB17 belongs to the thiazole/oxazole-modified microcin (TOMM) class of natural products. Invivo , the fullymodifiedMcbA peptide is liberated from the synthetase via proteolytic cleavage of a leader peptide by theTldDE protease, whose structure and mechanism were previously elucidated by the research group using data collected on beamlines 3 at Diamond Light Source. However, in a tldD deletion mutant, the modified peptide remains tethered to the complex. Through the attachment of an N-terminal hexahistidine tag to McbA, it was possible to affinity purify the whole complex when overexpressed in this deletion mutant. Following further purification, this sample was used for crystallisation trials. After optimisation of promising conditions, diffraction quality crystals were obtained through an iterative microseeding procedure. X-ray data to a maximum resolution of 1.85 Å were recorded on Diamond beamlines and the structure was resolved by the Single wavelength Anomalous Dispersion (SAD) method using a selenomethionine-substituted crystal. Prior to this study, the stoichiometry was assumed to be 1:1:1 for McbBCD, but the structure revealed an additional copy of McbB in the asymmetric unit, giving a composition of McbB 2 CD. Moreover, this assembly was closely associated with a two-fold crystallographic symmetry-related copy, largely through interactions between opposed McbC subunits, to yield a McbB 4 C 2 D 2 octamer (Fig. 2).The latter was confirmed as the biologically-relevant assembly through size exclusion chromatography. Within each half of the octamer, the pair of McbB subunits adopt different conformations and together form a clamp that tethers the leader peptide of the fully processed McbA peptide. Although, McbA is not fully resolved in electron density maps beyond the leader peptide, fragmentary electron density was apparent for a number of heterocycles, including the C-terminal oxazole group, bound at disparate locations in the complex, enabling speculation regarding the path taken by the peptide in the synthetase complex. The ATP-dependent heterocyclase activity is associated with the McbD subunits (Fig. 1), which lie at opposite ends of the octamer (Fig. 2). A structure with ADP and phosphate bound enabled the delineation of the active site, leading to a proposed mechanism involving the phosphorylation of a hemiorthoamide intermediate, with the C-terminal proline residue acting as a general base, which was supported by in vitro experiments with selected site- directed mutants. The FMN-dependent dehydrogenase activity resides within the McbC subunit (Fig. 1). Lying at the core of the complex (Fig. 2), each active site is comprised of amino acid residues from both McbC subunits. In addition to the tightly bound cofactor, the active site contains the terminal heterocycle of modified McbA, which stacks against the flavin moiety. Based on this arrangement, a mechanism involving the abstraction of a proton from the α-carbon of the azoline substrate by Lys201 and Tyr202 was proposed. However, the lack of a suitable general base in the vicinity of the N1 atom of FMN suggests that the cofactor is not fully reduced to FMNH 2 , but that the negative charge that develops on N1 could be stabilised by a salt bridge to the adjacent Arg233 to yield a hydroquinone anion. Despite the pair of McbC subunits at the core of the octamer being closely associated, it seems likely that each McbB 2 CD assembly functions Figure 1 : Sequence of McbA, the precursor for MccB17 biosynthesis. The leader peptide is indicated in green, and heterocyclisation sites are shown in red and yellow. Shown below are the two distinct catalytic activities of cyclisation and oxidation, associated with the McbD and McbC subunits, respectively. independently of the other. Previous work has shown that heterocycles are introduced sequentially from the N- to the C-terminus of the McbA precursor peptide, with each one being fully formed before the next 4 . This observation necessitates the peptide to repeatedly flip between the heterocyclase (McbD) and dehydrogenase (McbC) active sites, which are separated by a distance of around 40 Å, whilst remaining tethered to the peptide clamp formed by the McbB subunit pair (Fig. 3). Through this work, facilitated by access to the Diamond MX beamlines, almost 30 years of study on the biosynthesis of MccB17 has been reconciled, by shedding light on how the activities of the heterocyclase and dehydrogenase are temporarily and spatially coordinated during TOMM modification. More broadly, since oxazoles and thiazoles are found in a wide variety of bioactive natural products 5 , this knowledge could enable the decoration of any given peptide with azoles following well-determined rules, towards the creation of new therapeutics. References: 1. Collin F. et al. Exploiting bacterial DNA gyrase as a drug target: current state and perspectives. Appl. Microbiol. Biotechnol. 92 , 479-497 (2011). DOI: 10.1007/s00253-011-3557-z 2. Collin F. et al. The microbial toxin microcin B17: prospects for the development of new antibacterial agents. J. Mol. Biol. 431(18) , 3400- 3426 (2019). DOI: 10.1016/j.jmb.2019.05.050 3. Ghilarov D. et al. The origins of specificity in the microcin-processing protease TldD/E. Structure 25(10) , 1549-1561.e5 (2017). DOI: 10.1016/j.str.2017.08.006 4. Milne J. C. et al . Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17. Biochemistry . 38(15) , 4768-4781 (1999). DOI: 10.1021/bi982975q 5. Walsh C. T. et al. Three ring posttranslational circuses: insertion of oxazoles, thiazoles, and pyridines into protein-derived frameworks. ACS Chem. Biol. 7(3) , 429-442 (2012). DOI: 10.1021/cb200518n Funding acknowledgement: National Science Centre, Poland (UMO- 2015/19/P/NZ1/03137); EU Horizon 2020 (665778); RFBR-Royal Society International Exchanges Scheme (KO165410043/IE160246); BBSRC BB/ J004561/1 and BB/P012523/1). Corresponding author: Prof David Lawson, John Innes Centre,
[email protected] Figure 2: The full McbB 4 C 2 D 2 octamer in cartoon representation as viewed down the crystallographic two-fold axis (indicated by the black symbol) with individual subunits labelled, where those belonging to the right-hand asymmetric unit are preceded by the hash (#) symbol. Two copies of the leader peptide are shown in green cartoon representation, and as van der Waals spheres: ADP (blue), phosphate (magenta), FMN (cyan) and bound heterocycles (red). Figure 3: Schematic representation summarising the sequence of events leading to the production of mature MccB17. Firstly, the McbBCD synthetase binds the McbA precursor peptide via its leader peptide, between the two copies of McbB (McbB1 and McbB2) and then sequentially adds heterocycles. This involves the repeated shuttling of the precursor between McbD and McbC active centres. After the final modification, cleavage of the leader peptide by the TldDE protease yields the mature antibiotic, which is then released from the complex.