23 22 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 Macromolecular Crystallography Group Beamline I23 Investigating the role of metal ions in highly structured bacterial S-layers Related publication: Herdman, M., von Kügelgen, A., Kureisaite-Ciziene, D., Duman, R., el Omari, K., Garman, E. F., Kjaer, A., Kolokouris, D., Löwe, J.,Wagner, A., Stansfeld, P. J., & Bharat, T. A. M. High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells. Structure 30 , 215-228.e5 (2022). DOI: 10.1016/j.str.2021.10.012 Publication keywords : Caulobacter crescentus ; S-layer; bacteria; Cryo-EM; cryo-ET; Fluorescence microscopy; Long-wavelength X-ray diffraction; Metal-ion-binding proteins S urface layers (or S-layers) are present in most prokaryotic cells. They’re made from a diverse family of proteins capable of forming two-dimensional arrays that encompass the entire cell. Their ability to formhighly structured lattices with regular symmetry and self- healing properties makes them an attractive target for synthetic biology and studies of fundamental cell biology. To harness S-layers for synthetic biology, we need a better understanding of how they form, how they bind to the cell surface, and how they are maintained. One of the most commonly observed features is the utilisation of metal ions by S-layers for assembly and binding to the cell surface. Researchers used the Long-Wavelength MX beamline (I23) to study how bacterial cells (from Caulobacter crescentus ) use Ca 2+ ions bound to the constituent S-layer protein, RsaA, to facilitate the formation of the S-layer. Their study was highly interdisciplinary and used a range of methods frommicroscopic to near-atomic resolution. S-layershavearangeofapplications insyntheticbiology, asprotein-expressionplatformsorvaccinedevelopment systems, inbioremediation, and several other fields. S-layer biology is also a growing fieldwith implications for fundamental prokaryotic cell biology. This study provides insight into how S-layers optimise their own biogenesis using available metal ions in the environment, which can inform the design of synthetic two-dimensional sheets with long-range order up to the micron level. Surface layers (or S-layers) are a group of proteinaceous, two-dimensional lattices that constitute the outermost layer of many prokaryotic cell envelopes 1 . They are found in both Gram-negative and Gram-positive bacteria as well as in the enigmatic and poorly studied archaea 1 . S-layer proteins (SLPs) are highly diverse in sequence and are typically the highest-copy number proteins in expressing cells, making them one of the most ubiquitous protein groups in nature 1 . Despite the lack of obvious sequence homology, SLPs have several features that have been observed across multiple species and domains of life 1 . Of particular interest is the ability of S-layers to utilise environmentally available metal ions to facilitate their folding and retention along the cell surface, to form a regularly arranged lattice 1 . Due to this ability to self-assemble, S-layers have attracted interest due to their potential for applications in synthetic biology and for their importance in fundamental prokaryotic cell biology. This study 2 aimed to investigate S-layer biogenesis in the model organism Caulobacter crescentus , a Gram-negative bacterium with a hexagonal S-layer comprised of a single protein called RsaA. C. crescentus requires a high concentration of Ca 2+ (500 µM) for optimal growth, and previous research has suggested that this high Ca 2+ requirement is linked to the dependence of the S-layer on Ca 2+ ions for polymerisation. Lower concentrations of Ca 2+ result in slower growth and triggers S-layer shedding from the cell surface. Using an engineered strain of C. crescentus expressing a SpyTag-peptide, the researchers irreversibly labelled the S-layer using SpyCatcher proteins conjugated with different fluorescent proteins 2 . With this approach, they were able to directly track S-layer assembly and retention as a function of Ca 2+ concentration in themedium (Fig. 1A-G). The researchers observed that without ample Ca 2+ , the S-layer was lost from the cell surface, confirmed using electron cryotomography (cryo-ET), indicated by large gaps along an aberrant S-layer (Fig. 1H-I). The locations of 22 metal binding sites in RsaA were previously proposed using X-ray crystallography of S-layer sheets 3 . Molecular dynamics (MD) simulations of the RsaA hexamer revealed that Ca 2+ ions at these locations stabilised the hexameric structure of RsaA. However, two positions in RsaA, (positions 17 and 18), showed unstable Ca 2+ binding in simulations. To unambiguously confirm these previous predictions from crystallography and MD, the in vacuum beamline I23 at Diamond Light Source was utilised. This beamline provides access to measurements below and above the X-ray absorption edges of calcium (K edge: 4.0381 keV or 3.0704 Å) and potassium (K edge: 3.6074 keV or 3.4369 Å) 4 , not available anywhere else in the world. Using measurements at the I23 beamline, the researchers were able to identify and locate the positions of the Ca 2+ ions in the RsaA S-layer lattice 2 . Anomalous diffraction experiments showed that each monomer of the C-terminal domain of RsaA is bound to 17 Ca 2+ ions, identified using datasets collected using X-rays with energies of 4.10 and 3.95 keV (Fig. 2), confirming their previous results. Furthermore, as suggested by MD simulations, positions 17 and 18 did not contain Ca 2+ , satisfyingly confirmed by direct experiments. Position 17was bound to a K + ion, while position 18was neither Ca 2+ nor K + (Fig. 2). Thus, long-wavelength anomalous X-ray diffraction experiments allowed them to confirm predictions made from their previous X-ray data 3 and fromMD simulations, and helped them locate and identify metal ions in the RsaA lattice, of clear importance to the cell biology of C. crescentus . Finally, to complement Ca 2+ identification at I23, theywere also able identify Ca 2+ binding sites in other parts of the S-layer using Ho 3+ substitution of Ca 2+ ions in our previously described S-layer complex with lipopolysaccharide 5 . Ho 3+ has a high propensity for replacing Ca 2+ ions and cryo-EM single particle analysis of the complex produced a 4.3 Å resolution map that revealed positions of Ho 3+ substitutions. When taken together, the cryo-EM and long-wavelength studies have allowed the researchers to confirm the positions of 108 Ca 2+ ions in the RsaA S-layer hexamer, demonstrating the power of modern structural biology to bring atomic level insight into cell biological phenomena at the micron scale (Fig. 3). References: 1. Bharat, T. A. M. et al . Molecular logic of prokaryotic surface layer structures. Trends in Microbiology 29 , 405–415 (2021). DOI: 10.1016/j.tim.2020.09.009 2. Herdman, M. et al . High-resolution mapping of metal ions reveals principles of surface layer assembly in Caulobacter crescentus cells. Structure 30 , 215- 228.e5 (2022). DOI: 10.1016/j.str.2021.10.012 3. Bharat, T. A. M. et al. Structure of the hexagonal surface layer on Caulobacter crescentus cells. Nature Microbiology 2 , 17059 (2017). DOI : 10.1038/ nmicrobiol.2017.59 4. Wagner, A. et al . In-vacuum long-wavelength macromolecular crystallography. Acta Crystallographica Section D Structural Biology 72 , 430–439 (2016). DOI: 10.1107/S2059798316001078 5. Von Kügelgen, A. et al. In situ structure of an intact lipopolysaccharide- bound bacterial surface layer. Cell 180 , 348-358.e15 (2020). DOI: 10.1016/j. cell.2019.12.006 Funding acknowledgement: M.H. is supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC, grant number BB/M011224/1). T.A.M.B. is a recipient of a Sir Henry Dale Fellowship, jointly funded by theWellcome Trust and the Royal Society (202231/Z/16/Z). T.A.M.B would like to thank the Vallee Research Foundation, the Leverhulme Trust, and the John Fell Fund for support. Corresponding authors Matthew Herdman, University of Oxford,
[email protected] Dr. Tanmay Bharat, University of Oxford,
[email protected] Figure 1: C. crescentus cells expressing RsaA-467-SpyTag grown in M2G media containing (A) 500 µM; (B) 250 µM; (C) 100 µM; (D) or no additional CaCl 2 , with incubation with SpyCatcher-mRFP1; Controls included (E) 1000 µM MgSO 4 and no CaCl 2 and with SpyCatcher-mRFP1 and (F) cells grown without SpyCatcher-mRFP1. mRFP1 signal is strongest in cells grown in 500 µM CaCl 2 with a clear reduction at lower concentrations (scale bar: 10 µm); (H) Average cell intensity and standard deviation was quantified using ImageJ (n = 50); Slices through tomograms of cells grown in (H) 500 µM CaCl 2 or (I) 100 µM CaCl 2 M2G media, showing S-layer disruption at lower CaCl 2 concentrations. Figure 2: (A) The RsaACTD hexamer showing proposed metal-ion-binding sites (yellow spheres); Chains are identified as α-ζ. Densities in anomalous difference maps collected at X-ray energies of 4.1 and 3.7 keV represent calcium (green mesh) and potassium (magenta mesh) respectively. All maps are displayed at a contour level of 4.5 σ. Positions 1-16 and 19 are coordinated with Ca 2+ densities, with few exceptions. Position 17 is coordinated with a K + ion in our three-dimensional crystals in all RsaA monomers except δ, while 18 was consistently empty; (B) Magnified chain α of the RsaA hexamer (blue); (C-E) Magnified views of RsaACTD in A. Figure 3: Surface model of the RsaA hexamer (RsaACTD displayed in blue, RsaANTD in purple) in (A) top view and (B) bottom view with associated metal ions as confirmed by experiments. Confirmed Ca 2+ ions (positions 1-16, 19 and 21) are shown in green, metal-binding sites with unassigned or no associated ions are displayed in red, i.e. positions 17 (K + ), 18 (possibly Mg 2+ ), 20 and 22 (probably Ca 2+ ); (C-D) Orthogonal side views. Scale bar: 50 Å .