Diamond Annual Review 2021/22

34 35 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 2 1 / 2 2 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 2 1 / 2 2 Understanding howbacteria attack each othermay help to develop newdrugs Related publication : Ghilarov, D., Inaba-Inoue, S., Stepien, P., Qu, F., Michalczyk, E., Pakosz, Z., Nomura, N., Ogasawara, S.,Walker, G. C., Rebuffat, S., Iwata, S., Heddle, J. G., & Beis, K. Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides. Science Advances 7, (2021). DOI: 10.1126/sciadv.abj5363 Publication keywords : Antibacterial peptide; SLiPT transporter; ABC transporter; Glutamate ladder; Proton driven transporter B acterial species compete for access to nutrients. Many use biological weapons that target and kill closely related species. These antibacterial compounds include antibacterial peptides such as microcins which do not damage the membrane integrity (essential for bacterial survival) but inhibit specific proteins. Previous investigations have shown that while these peptides are structurally diverse, they use the same transporter (SbmA/BacA) to enter the cell. Researchers wanted to understand this process by determining its structure andmolecular mechanism. Their aimwas to gain insights into the molecular mechanism bacteria use to hijack the SbmA/BacA transporter. This information could then be used to develop novel therapies that can use the same uptake process. The teamused Cryo ElectronMicroscopy at the electron Bio-Imaging Centre (eBIC) at Diamond Light Source to determine the structure of the transporterin a lipid-like environment that mimics the native environment of the transporter. Determining the structure has allowed the team to identify a new family of transporters, which they have called SbmA-like peptide transporters (SLiPTs). Interestingly, the structures show similarities with another family of transporters that are usually involved in multidrug resistance, but the role of SLiPTs is to internalise multiple substrates. The scientists believe there is an evolutionary link between the two families that could allow us to understand how resistance evolved. Microcins are small antibacterial peptides (<10 kDa) produced by enteric bacteria and target closely related species. Many microcins are known to be ribosomally-synthesised post-translationally modified peptides (RiPPs), which are produced as linear precursors and require post-translational modification in order to become biologically active (Fig. 1). Since these peptides do not damage the membrane, but instead have intracellular targets ( e.g. DNA gyrase or RNA polymerase), they must penetrate the outer and inner membrane for internalisation. MccB17 crosses the outer membrane via the porin OmpF, whereas MccJ25 uses the ferrichrome receptor FhuA 1 . However, when it comes to crossing the inner membrane, despite their structural differences, most microcins utilise a common transporter, SbmA. The E. coli SbmA (EcSbmA) was identified as essential for sensitivity to thiazole-modified RiPP antibiotic MccB17 and subsequently was confirmed as the most important determinant of sensitivity to several structurally unrelated NPs from Gram-negative bacteria, including the lasso peptide MccJ25, the azole-modified peptide klebsazolicin (KLB) and the NRP-polyketide azole-containing antibiotic bleomycin (Fig. 1). While the exact natural function of EcSbmA is not established, it is a virulence factor in an avian pathogenic E. coli (APEC), while its Brucella abortus ortholog has been shown to be required for chronic intracellular infection in a mouse model. Interestingly, the E. coli SbmA is isofunctional with its homolog BacA from Sinorhizobium meliloti that is indispensable for the nitrogen-fixing symbiosis with the legume host; BacA enables the chronic intracellular infection of root nodule cells, by transporting nodule-specific defensin-like cysteine-rich peptides. To establish the molecular mechanism of peptide transport and substrate Biological Cryo-Imaging Group eBIC promiscuity of EcSbmA/SmBacA, the authors determined the cryo–Electron Microscopy (cryo-EM) structure of both SbmA and BacA proteins in a lipid bilayer mimetic environment, using proteins reconstituted in nanodiscs (NDs), at 3.2 and 6 Å local resolution, respectively 2 . SbmA is a homodimer consisting of eight Transmembrane (TM) helices per protomer. SbmA consists of two TM0 domains and a coreTMdomain (TMD), which comprises 12TMhelices (Fig. 1). The overall structure of TMD notably resembles the TMD of ABC transporters (exporters), such as MsbA (lipid A flippase) from E. coli , Sav1866 (multidrug resistance) from Staphylococcus aureus , and Rv1819c (cobalamin transporter) from M. tuberculosis . Despite these similarities, the structural organisation of SbmA has not been observed in secondary transporters before. Therefore, SbmA defines a new fold for secondary transporters, named SbmA-like peptide transporter (SLiPT) fold. The SbmA dimer interface is formed betweenTMs 1 and 2 and 5 and 6 fromone protomer and the equivalent ones from the second; TMs 1 and 2 form intermolecular contacts with TMs 5 and 6, and TMs 5 and 6 form intermolecular contacts with TMs 1 and 2. The TM0 domain of SbmA is a novel feature of SLiPT transporters, which is not present in any other known transporter structure. SbmA is trapped in an outward-open conformation ready to bind antibacterial peptides for internalisation. Considering the important biological role of BacA in symbiosis, and especially that EcSbmA is isofunctional with the SmBacA, its structure was also determined. The BacA reconstruction displays the same overall fold as SbmA; the SbmA structure can fit in the BacA map without any structural changes, suggesting that BacA also adopts a very similar fold (Fig. 1). To probe the mechanism of proton translocation, SbmA and BacA were reconstituted in liposomes; transport could only be initiated in the presence of both a proton gradient (induced by valinomycin) and the substrates bleomycin or MccB17. The SbmA cavity is lined with conserved Glu residues, forming a “glutamate ladder,” suggesting a relay path for proton movement (Fig. 2). To probe their role in coordinating proton translocation, the glutamates were mutated to alanines and measured their substrate transport activity. All Glu-to-Ala mutations essentially abolished substrate transport. In light of the structural and functional data, a mechanism for antibacterial peptide import by the SLiPT transporters SbmA and BacA has been proposed; in brief, SbmA adopts an outward-open conformation with its cavity accessible to both peptide and proton. Binding of a proton in Glu203, with subsequent movement along the glutamate ladder, and simultaneous peptide binding in the open cavity induce either a transient or a stable occluded state (Fig. 3). An inward-facing open conformation can be formed by movement of TMs 4 and 5 away from TMs 4 and 5. All fully resistant mutants are found along TM5, suggesting its important role in substrate translocation associated with conformational changes along the transport cycle. In the inward-open conformation, the antibacterial peptide and proton are released in the cytoplasm, and the transporter resets in the outward-open conformation. References: 1. Mathavan, I. et al. Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides. Nature Chemical Biology 10 , 340342 (2014). DOI: 10.1038/nchembio.1499 2. Ghilarov, D. et al. Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides. Science Advances 7, (2021). DOI: 10.1126/sciadv.abj5363 Funding acknowledgement: Biotechnology and Biological Sciences Research Council grant BB/H01778X/1 (KB), Team program of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund TEAM/2016- 3/23 (JGH, PS), National Science Centre (NCN, Poland) grant no. 2016/21/B/ CC1/00274 (OPUS 11) (ZP, DG and JGH) and 2019/35/D/NZ1/01770 (SONATA 15) (ZP, EM and DG), Chinese Scholarship Council Scheme (FQ) Corresponding author: Konstantinos Beis, Imperial College London, kbeis@imperial.ac.uk Figure 1: Cryo-EM structure of SLiPT transporters; (a) Antibiotic peptide substrates of SLiPT transporters; (b) EMmap of SbmA-FabS11-1-ND complex. The two maps contoured at different levels are overlaid to represent both high-resolution (SbmA) and low-resolution (ND) parts; (c) Cartoon representation of the SbmA structure. SbmA is a homodimer consisting of two TM0 domains and a core TM domain; (d) The low-resolution EMmap of BacA-ND shows fold conservation in the SLiPT transporter family; the SbmA structure has been fitted within the volume. Figure 2: A unique pathway for protons; (a) A cartoon representation of SbmA and glutamates, forming the glutamate ladder; (b) Mutations along the glutamate ladder have a detrimental effect in the movement of protons and the substrate. Figure 3: Molecular mechanism of SLiPT transporters.