The molecular architecture of Lactobacillus S-layer: Assembly and attachment to teichoic acids

Significance S-layer proteins (SLPs) are self-assembling, crystalline proteins coating the cell surfaces of many prokaryotes. This study presents experimental atomic resolution structures of lactobacilli SLPs, deriving functional insight into key probiotic Lactobacillus strains. The structures of SlpA and SlpX proteins highlight the domain swapping critical for SlpX integration, particularly in response to environmental stress. Two binding regions are identified as crucial for attachment of the S-layer to (lipo)teichoic acid. The structure of assembled S-layer provides a foundation for employing (designed) SLPs as a therapeutic agent in the treatment of inflammatory diseases. Additionally, it opens broad avenues for the use of SLPs in vaccine development and in crafting nanostructures with tailored properties, including those designed for targeted drug delivery.


Supporting Information Text
Evaluation of proposed SlpA assembly with published mutagenesis data.The proposed selfassembly model is further reinforced using the results of the published mutagenesis data reported by Smit et al. 2002 1 .These experiments quantify the effect of inserted loops between 8aa and 19aa in length on the in vitro ability of SlpA to assemble in crystalline form.Our assembly model proposed in this study is supported by results with mutations located in contact regions or binding sites and results with mutations located in accessible regions without close contact with other domains (Figure S9).At the binding site between SlpA_I and its N-terminus, 3 mutation locations were tested for their effect on the S-layer assembly.These mutations are located in the SlpA binding pocket and the N-terminal tail.All 3 mutations considerably diminish or prevent the formation of crystalline assemblies, thus suggesting a crucial role of this binding site for layer assembly and confirming the assembly model presented in this study.In the contact region between SlpA_I and SlpA_II of distinct neighboring monomers, 3 mutations were tested for their effect on the S-layer assembly.These mutations either do not assemble or show only to a lower degree of assembly.Two mutations, one in the intramolecular contact region between domain SlpA_I and SlpA_II and one near the linker between both domains, inhibit crystal layer formation.The second mutation likely affects the linker between both domains, resulting in a different domain conformation that hinders the S-layer formation.Both observations are consistent with the proposed assembly model.Insertion mutations located at the surface of SlpA_I without close contact with other domains were compatible with layer formation; thus, these results support the proposed assembly model.

Methods
Figure S1.AlphaFold multimer prediction of full-length SlpA.A) Assembly of 6 full-length SlpA (SlpA_III facing downwards not shown) molecules as predicted by AlphaFold multimer 4,5 .The Nterminus propagates the assembly in one direction and the dimer formation of SlpA_II in the other direction.B) Assembly of 6 full-length SlpA (SlpA_III facing downwards not shown) molecules as predicted by AlphaFold multimer without the first 16 N-terminal residues.An alternating SlpA_I -SlpA_I and SlpA_II -SlpA_II interface is predicted with the same interactions as in the crystal structures.The angle (flexible linker) between SlpA_I and SlpA_II is adjusted and not the same as in the fully assembled model.

Figure S2 .
Figure S2.Self-assembly mutation data derived from Smit et al. 2002 mapped on the proposed SlpA_ac assembly model.Assembly of crystallization domains (SlpA_ac_I, SlpA_ac_II), one SlpA_ac•SlpA_ac dimer is visually highlighted compared to surrounding neighbors; linkers between domains are shown as dashed lines.The locations of mutations are colored in terms of compatibility with assembly formation according to Smit et al. 20021 .Blue: mutations compatible with assembly and a high degree of assembly (25%-28%).Cyan: mutations compatible with assembly and low degree of assembly (19%-23%).Red: mutations not compatible with layer formation.The amino acid positions where the additional linker is inserted are labeled with numbers.

Figure S3 .
Figure S3.Comparison between simulated and available experimental projection maps.(A) simulated and (B) experimental projection maps of the full-length SlpA assembly layer.(C) simulated and (D) experimental projection maps, including only the SlpA assembly domains (SlpA_ac_I and SlpA_ac_II).Experimental projection maps are reported in Smit et al. 20016 .Simulated projection maps, based on the assembly model proposed in this study, were calculated using pdb2mrc7 in ChimeraX8,9

Figure
Figure S4.N-terminal cleft is highly conserved among different lactobacilli.Sequence alignment of the N-terminal residues of L. acidophilus, L. amylovorus, L. helveticus, L. gallinarum, and L. crispastus.Structures of SlpA_ac (7QLE) and SlpA_amy (8Q1O) are determined by X-ray crystallography, and structures of SlpH_hel, SlpA_gal, and SlpA_cri are calculated with AlphaFold multimer.The highlighted isoleucine (yellow) is conserved in all five species and positioned within the next molecule's cleft.The cleft is shown in a mesh colored from red (hydrophobic) to blue (hydrophile).Cavities were calculated using CavMan (Innophore GmbH) 10,11 .

Figure S5 .
Figure S5.The primitive unit cell of the proposed SlpA assembly.Black: Unit cell with a = 118 Å, b = 53 Å, γ = 102° as reported by Smit 2001 6 and Purple: a = 119 Å, b = 69 Å, γ = 132° as shown as one tile composed of a dimeric subunit as in Figure 4.Both tiles have the same area.

Figure S7 .
Figure S7.Conserved TAB-domain with bound phosphate.A) Alignment of the internal homodimer of N-terminal (ochre) and C-terminal (orange) SlpA motif.For SlpX_ac_III, an alignment of the additional SlpA motiv of domain II (grey) is shown.B) Alignment of N-and C) C-terminal SlpA motif of SlpA_ac_III (orange), SlpA_amy_III (ochre) and SlpX_ac_III (grey).

Figure S8 .
Figure S8.Conserved TAB-domain motif across different proteins in Lactobacillus.P38059, L. helveticus; A0A0PECX7L, L. gallinarum; Q09FM2 L. crispatus.Residues highlighted in yellow are conserved and involved in TAB-domain binding motif and correspond to the residues shown in Figure 5. Residues indicated with a star show only backbone interaction.The asterisk (*), colon (:), and semicolon (.) indicate fully conserved residue, conserved between groups of strongly similar properties and conserved between weakly similar properties, respectively.

Figure S9 .
Figure S9.CSP analysis of SlpA_amy_III and GroP trimer.A: The presented graph shows the calculated d-values for each residue.Residues with high d-values are highly influenced by ligand interaction and are either located in or close to the binding pocket or experience conformational changes.

Figure S10 .
Figure S10.Analysis of SlpA_amy_III dynamics via NMR relaxation experiments.For each residue { 1 H}-15 N-hetero NOE values and rotational correlation coefficients were determined.T1 and T2 relaxation data were used for the calculation of rotational correlation coefficients.Data reveals increased dynamics only at the N-terminal region, whereas the rest of the protein exhibits high { 1 H}-15 N-hetero NOE values indicating a rigid stable fold of SlpA_amy_III.The average rotational correlation time of SlpA_amy_III in solution is 10 nsec corresponding to a well-structured 21 kDa protein (see 'Methods').The SlpA_amy_II construct has an MW of approximately 16 kDa.

Figure S11 .
Figure S11.CSP derived HADDOCK models of SlpA_amy_II and GroP pentamer.For each binding site a separate HADDOCK run was performed.Models of lowest HADDOCK-scored clusters for binding site 1 are shown in (A), for binding site 2 in (B).

Figure S12 .
Figure S12.Putative N-glycosylation sites (Asn -X -Ser/Thr) of SlpA_ac.The possible Nglycosylation sites of SlpA_ac are depicted in red/blue with the sticks representation for the respective asparagines.Sites near the interaction surface between SlpA_ac monomers are shown in detail and colored in red, surface exposed sites are colored blue.The sequence of SlpA_ac is shown with the highlighted glycosylation sites.

Figure S13 .
Figure S13.Schematical overview of the cell wall composition of Lactobacillus.S-layer composed of SlpA is shown as the outermost layer.The domains involved in the self-assembly are shown in green and the TAB-domain is shown in yellow.Putative glycosylation of the S-layer is omitted in this figure.Lipoteichoic acids anchored in the cytoplasmic membrane are shown in blue and peptidoglycon-anchored wall teichoic acids are in red.Membrane proteins are colored in violet and in light blue.

Table S1 .
List of to date available experimental structures of assembled S-layers and SLP fragments.

Table S2 .
Constructs used in this work.Residue numbering includes signal sequence.

Table S3 .
Primers used for cloning.All primers were either designed with the NEBaseChanger, SerialCloner, or by hand.

Table S4 .
Screens and final conditions used for crystallization of all fragments.

Table S5 :
Crystallographic table for data collection and refinement statistics.Values in parentheses are for the highest-resolution shell.

Table S7 .
Calculation of dissociation constants.Kd for SlpA_amy_III and GroP trimer by NMR according to formula in the 'Materials and Methods' section resulting in a mean Kd value of 3.5 mM.

Table S8 .
Calculation of dissociation constants.Kd for SlpA_amy_III and GroP pentamer by NMR according to formula in the 'Materials and Methods' section resulting in a mean Kd value of 0.5 mM.

Composition of SlpA layer model based on experimental crystal structures.
This movie shows how the proposed SlpA layer model comprises experimental crystal structures.The starting point is the experimental SlpA_ac_I crystal structure, which consists of coils of SlpA_I • SlpA_I dimers connected by their N-termini.The first part of the movie shows how these SlpA_I • SlpA_I dimer chains can be uncoiled and placed on a 2D plane.The movie's second part shows how the SlpA_I •SlpA_I dimer chains are complemented by SlpA_II and SlpA_III domains (shown in 2 SpA_motifs) and how they assemble to the complete SlpA layer.