Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein Jaslyn E. M. M. Wong1, Husam M. A. B. Alsarraf1,*, Jørn Døvling Kaspersen2, Jan Skov Pedersen2, €l Blaise1 Jens Stougaard1, Søren Thirup1 and Mickae 1 Department of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling, Aarhus University, Denmark 2 Department of Chemistry, Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Denmark
Keywords autolysin; chitin; lysin motif (LysM) domain; NlpC/P60 endopeptidase; peptidoglycan Correspondence M. Blaise, Department of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling, Aarhus University, Gustav Wieds Vej 10, Aarhus 8000, Denmark Fax: +45 8612 3178 Tel: +45 8715 5524 E-mail: [email protected] *Present address: Statens Serum Institut, Copenhagen University, Artillerivej 5, Copenhagen S, 2300, Denmark (Received 30 June 2013, revised 12 December 2013, accepted 17 December 2013) doi:10.1111/febs.12698
Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these b,1–4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydratebinding site in LysM-containing proteins is conserved across phyla.
Introduction The bacterial cell wall consists of a rigid layer of peptidoglycan (PGN) that maintains the integrity of the bacterial cell and confers protection against environmental stress. PGN is a polymer of GlcNAc and N-acetylmuramic acid (MurNAc) arranged in a three-dimensional network crosslinked by peptide chains that connect MurNAc moieties. Although PGN is rigid, it also has to be highly dynamic to allow the bacterial cells to elongate and separate during growth and division [1]. Numerous enzymes grouped under the
family of autolysins are involved in PGN degradation and remodelling, thereby contributing significantly to PGN structural dynamics. These enzymes have diverse activities: glycosidases are lysozyme-type enzymes that cleave the carbohydrate chain of PGN, whereas endopeptidases, amidases and carboxypeptidases hydrolyse the peptide stem of PGN at different positions. Among the autolysins, NlpC/P60 D,L-endopeptidases play a crucial role in the remodelling of PGN. Most D, L-endopeptidases cleave the peptide chain of PGN
Abbreviations CwlS, cell wall-lytic enzyme associated with cell separation; GFB, gel filtration buffer; GlcNAc, N-acetylglucosamine; LysM, lysine motif; MST, microscale thermophoresis; MurNAc, N-acetylmuramic acid; PGN, peptidoglycan; SA, specific activity; SAXS, small-angle X-ray scattering.
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between the second and third residues. In bacteria, NlpC/P60 enzymes are modular proteins composed of a catalytic domain, named NlpC/P60, that is very often associated with an additional domain(s) predicted to be involved in PGN binding. Indeed, the SH3b, cholinebinding domain and lysin motif (LysM) [2,3] domains are very often found in NlpC/P60 endopeptidases. The LysM domain, a small protein domain of ~ 50 residues, has been defined as a carbohydrate-binding domain with affinity for GlcNac polymers [4]. In bacteria, the LysM domain is often associated with proteins involved in PGN synthesis or remodelling, and several LysM domains are frequently found in these enzymes [4]. Few biochemical studies have demonstrated direct binding between LysM domains and PGN, and the binding studies have typically been conducted with insoluble PGN for affinity pulldown assays. Rigorous determination of the substrate affinity of bacterial LysM domains has not been achieved, as biochemical studies are hampered by the properties of PGN. PGN is insoluble when extracted from bacteria, and PGN oligomers are difficult to synthesize or purify in large amounts. Thus, it is not trivial to perform PGN-binding experiments to assess binding affinities. Illustrating this complexity, binding of the LysM-containing protein Lactococcus lactis AcmA to bacterium-like particles and PGN was shown by steady-state fluorescence, but it was not possible to determine affinity constants [5]. In summary, despite recent progress in studying bacterial LysM-containing proteins, several questions
Binding studies on bacterial LysM domain
remain unanswered. Notably, these concern the affinities for their ligands, the correlation between the number of LysM modules and the affinity for carbohydrates, the ligand recognition specificity, and, finally, the binding site localization. In this study, we addressed these questions by using biochemical approaches to study cell wall-lytic enzyme associated with cell separation (CwlS), which is involved in cell separation during division of Bacillus subtilis [6]. CwlS is an NlpC/P60 D,L-endopeptidase, possessing four LysM domains in the N-terminus followed by a C-terminal NlpC/P60 catalytic domain [2]. It is therefore an appropriate protein model for investigating the individual and cooperative role(s) of LysM domains in carbohydrate binding.
Results Expression and purification of CwlS To investigate the influence of LysM modules on the binding affinity of CwlS, we first purified the CwlS fulllength protein (FL) and CwlS catalytic domain (Cata), by using the Escherichia coli expression system. Subsequently, we expressed and purified a series of protein derivatives (Fig. 1). The N-terminally to C-terminally LysM-truncated mutants lack one (3LysM-Cata), two (2LysM-Cata) or three (1LysM-Cata) of the LysM domains in the N-terminus; the C-terminally to N-terminally LysM-truncated proteins mutants lack the catalytic domain (4LysM) plus one (3LysM), or two
Fig. 1. Schematic of the constructs used in this study. The LysM domains are shown in grey, and the catalytic domain in light grey. The residue numbering is indicated. The mutations of the LysM domain are indicated by ‘A’ followed by a number giving the position.
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(2LysM) LysM domains in the C-terminus. To examine the binding capacity of individual LysM domains, single LysM domains (1LysM_1, 1LysM_2, 1LysM_3 and 1LysM_4) numbered 1–4 from the N-terminus were also expressed and purified. Finally, to assess the carbohydrate recognition mode and to further supplement the single LysM studies, we used an Ala scanning mutagenesis approach to express and purify a series of binding site variants (FL_Mut and FL_Mut1, FL_Mut1_2, FL_Mut1_3, and FL_Mut1_4). All proteins could be purified to ~ 95% purity and appeared as a single homogeneous peak in size exclusion chromatography, strongly indicating that they are monomeric and folded. The number of LysM modules affects the endopeptidase activity of CwlS We first wanted to determine whether deletion of LysM domains might influence the endopeptidase activity and thereby the cell wall-degrading capacity of CwlS. For this purpose, purified proteins were added to exponentially growing B. subtilis cultures, and cell densities were measured. Addition of purified full-length protein resulted in decreased cell density over time with a specific activity (SA) of 99 U (where U is defined as lAmin 1lg 1 of enzyme; see Experimental procedures), demonstrating that the purified protein was active (Fig. 2A). In this assay, 3LysM-Cata seemed to be less active, with an SA of 68 U. The loss of activity was even more pronounced when two or three LysM domains were deleted (2LysM-Cata, SA = 33 U; 1LysM-Cata, SA = 28 U). Moreover, the cell density did not decrease over time when all LysM domains were removed, suggesting an absence of endopeptidase activity on bacterial cell walls. The number of LysM modules affects PGN binding To assess whether the correlation of the decreased endopeptidase activity and decreased number of LysM modules was linked to the loss of PGN-binding capacity, we performed semiquantitative PGN pulldown assays (Fig. 2B,C). This assay exploits the insoluble nature of bulk PGN. Proteins binding to insoluble PGN will therefore coprecipitate with PGN and be found in the pellet after centrifugation. The PGN pulldown experiments showed that the full-length protein and 3LysM-Cata had similar binding capacities, as approximately 44% and 45% of the proteins were pelleted (Fig. 2C). However, binding strength appeared to decrease with the number of LysM modules present, as only 28%, 20% and 18% 1198
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of 2LysM-Cata, 1LysM-Cata, and the catalytic domain, respectively, bound to PGN (Fig. 2C). This suggests that the binding affinity correlates with the number of LysM modules, and that the catalytic domain has only low affinity for PGN. LysM domains bind PGN fragments in the lowmicromolar range To further characterize the affinity for PGN, we assessed the binding of CwlS proteins to different polymer lengths of commercially available PGN fragments (Fig. 3) purified from Staphylococcus aureus or B. subtilis. In this assay, microscale thermophoresis (MST) [7], a rather new technique that has been proven to be adequate for binding studies with aqueousphase carbohydrates, was used [8]. We first tested a soluble PGN tetramer from S. aureus and a soluble PGN trimer from B. subtilis (Fig. 3). Both were extracted from cell walls treated with glucosaminidase and endopeptidase, which leaves only GlcNAc–MurNAc disaccharides. The carbohydrate polymerization is maintained by the peptide chain, as the MurNAc– GlcNAc bond has been hydrolysed (Fig. 3). MST experiments showed that the full-length protein had an equilibrium dissociation constant (KD) of 10.1 lM for S. aureus PGN tetramer (Table 1). Interaction also occurred when shorter PGN fragments were measured in this assay, but the binding affinities of S. aureus PGN trimer and B. subtilis PGN trimer were approximately one order of magnitude lower than that of S. aureus PGN tetramer (Table 1). In addition, the affinity for B. subtilis PGN dimer was ~ 30-fold lower than that for S. aureus PGN tetramer (Table 1). This demonstrates that bacterial LysM domains have an affinity for PGN fragments in the low-macromolar range. Furthermore, our data suggest that LysM domains have higher affinity for longer carbohydrate polymers. LysM multiplicity increases affinity for PGN To investigate the influence of LysM modules on binding affinity, we utilized the protein set where LysM domains had been removed one by one from the N-terminus to the C-terminus (Fig. 1). The binding capacities of these proteins were tested in aqueous phase with MST. 3LysM-Cata had a similar affinity for S. aureus PGN tetramer as the full-length protein, considering that the KD value was ~ 9 lM. However, removal of two or three LysM domains influenced the binding to PGN. Indeed, binding to PGN was not detectable for either 2LysM-Cata, 1LysM-Cata, 1LysM_1, or 1LysM_4. In contrast, a protein derivative possessing FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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Fig. 2. (A) Endopeptidase activity assay. The graph shows the D600 nm of B. subtilis cell cultures after protein addition. The protein set used in this assay is described in Fig. 1. Lysozyme and buffer were used as positive and negative controls, respectively. (B, C) Semiquantitative pulldown experiments. (B) Control experiment in which no PGN was added to the protein, to verify that proteins were not precipitating on their own after centrifugation. (C) Gel and quantification table of the pulldown experiment results. P, pellet; SN, supernatant. The table represents an average of four independent pulldown experiments.
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only the four LysM domains (4LysM) had similar affinity as the full-length protein, as it binds S. aureus PGN tetramer with an affinity of 6.8 lM. This shows that the binding contribution of the catalytic domain is negligible. This conclusion is reinforced by the fact that binding of the catalytic domain alone to PGN fragments was not detectable (Table 1). Our experiments clearly indicate that LysM domains are crucial for protein binding to PGN, and that the contribution of the catalytic domain to substrate recognition is minor. FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
+ PGN 2LysM Cata P SN
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CwlS protein
Proportion in pellet
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0.44 ± 0.08
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0.45 ± 0.11
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0.17 ± 0.08
CwlS LysM domains do not discriminate between GlcNAc polymers and MurNAc–GlcNAc polymers In plants, a variety of membrane receptors containing LysM domains are involved in recognition of GlcNAc polymers. Although these receptors are very similar to each other, they distinguish between three related substrates – lipo-chitooligosaccharide, chitin polymers, or PGN – whose recognition must be specific, given that each of these carbohydrate polymers triggers a different
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HO HO HO
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Fig. 3. Structure of the PGN fragments used in this study. The arrows indicate that the MurNAc–GlcNac glycosidic bond is not preserved in these ligands. mA2 pm, meso-diaminopimelic acid.
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Table 1. CwlS binding constants for PGN ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; NT, not tested.
Full-length protein 3LysM-Cata 2LysM-Cata 1LysM-Cata Catalytic domain 4LysM FL-Mut
Staphylococcus aureus tetramer
Staphylococcus aureus trimer
Bacillus subtilis trimer
Bacillus subtilis dimer
10.11 2.08 9.07 1.73 ND ND ND 6.81 1.31 ND
173.20 59.80 170.40 33.50 NT NT NT 479.90 211.30 NT
121.02 40.18 187.30 73.00 ND NT ND 207.80 72.70 NT
356.90 98.10 134.40 20.60 NT NT NT 222.45 56.85 NT
plant response pathway [9–11]. They lead either to plant defence (for chitin and PGN) or to symbiosis (lipo-chitooligosaccharide). Following this line of reasoning, we tested whether a bacterial LysM protein could discriminate between PGN and chitin GlcNAc polymers. Our MST binding experiments showed that the fulllength protein had a KD of 4.7 lM for chitooctaose (Table 2). As seen with the PGN fragments, the affinity decreased when shorter chitin polymers were assayed; chitohexaose, chitopentaose and chitotetraose, respectively, had KD values of 20.4, 316.4 and 765.7 lM. To extensively investigate the binding properties of a multiple LysM bacterial protein, we assessed the chitin-binding capacities of all of the CwlS protein derivatives (Fig. 1). The affinity was similar for chitooctaose when the full-length protein, 3LysM-Cata and 2LysMCata were compared, but only weak binding was observed for 1LysM-Cata (Table 2). Interestingly, 1LysM_1, which did not bind PGN fragments, was shown to bind chitin polymers with a KD of 21.2 lM, which is similar to value for the single LysM domain from Pteris ryukyuensis chitinase-A [12]. We further tested the other LysM domains to assess their individual binding affinities. 1LysM_2 and 1LysM_3 had higher affinity for chitin oligomers than 1LysM_1 (9.6
and 7.6 versus 21.3 lM, respectively, for chitooctaose), whereas only weak binding of chitooctaose was detected with 1LysM_4. This indicates that the different LysM domains have different binding affinities. To further explore this hypothesis, the chitin-binding capacities of 3LysM (LysM1 to LysM3) and 2LysM (LysM1 to LysM2) were assessed. These proteins had affinities similar to those of 3LysM-Cata and 2LysMCata (Table 2). Together, these data indicate that LysM1, LysM2 and LysM3 have similar affinities, whereas LysM4 seems to bind carbohydrates only weakly, as observed in the PGN pulldown assay (Fig. 2C) and MST assay (Table 2). Congruent with the MST results observed for PGN fragments, the binding affinities of the single LysM domains gradually decreased with a reduction in the length of the chitin polymers (Table 2). Similarity between the CwlS LysM carbohydratebinding sites and other LysM domains Several groups have proposed different binding sites for the LysM domains from different phyla, on the basis of docking and molecular dynamics [13,14]. Likewise, NMR studies have suggested residues that might
Table 2. CwlS binding constants for chitin ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; NT, not tested; >, saturation could not be reached.
Full-length protein 3LysM-Cata 2LysM-Cata 1LysM-Cata Catalytic domain 4LysM 3LysM 2LysM 1LysM_1 1LysM_2 1LysM_3 1LysM_4
Chitooctaose
Chitohexaose
Chitopentaose
Chitotetraose
4.78 0.58 3.13 0.77 9.89 2.19 > 1089.40 ND 1.53 0.07 1.83 0.17 7.88 1.11 21.29 1.16 9.63 1.41 7.61 1.80 > 1312.55
20.40 11.63 18.06 ND ND 14.15 12.74 24.34 44.74 12.17 9.92 ND
316.45 275.60 563.70 ND ND 288.10 243.04 393.75 520.40 328.75 186.45 NT
765.75 582.65 ND ND ND 810.65 444.95 483.05 787.85 384.20 > 1410.55 NT
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2.31 3.88 2.95
1.08 1.26 2.12 5.60 1.84 3.01
65.15 52.50 119.50
18.70 30.25 61.75 66.30 61.05 41.45
123.75 146.55
74.05 51.45 27.25 69.95 83.50
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be involved [12,15]. More recently, the crystal structure of the Arabidopsis chitin receptor CERK1 ectodomain–chitin complex has shed light on the binding site of a plant LysM2 domain. The CERK1 structure shows that chitin is bound by hydrophobic, Van der Waals and hydrogen bonds. At the reducing end of the carbohydrate, three residues, Met127, Gln131, and Ala138, recognized the N-acetyl group of GlcNAc4 (Fig. 4) [16]. To determine whether there are any similarities between the CERK1 LysM2 and bacterial LysM binding sites, we made homology models of the four LysM domains of CwlS and superposed them on the LysM2 domain of the CERK1 crystal structure. These models predict that Ile48, Lys52 and Thr60 from LysM1, Val109, Lys113 and Asp121 from LysM2, Val178, Lys182 and Asp190 from LysM3 and Val246, Arg250 and Asp258 from LysM4 could possibly engage in
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hydrophobic and Van der Waals interactions with GlcNAc4 N-acetyl group (Fig. 4). To test this hypothesis, we replaced these three residues with Ala in the four LysM domains, and determined the binding capacity of this protein derivative. These mutations are not expected to totally abolish the binding, because, in the CERK1 crystal structure, GlucNac residues 1–3 are also tightly bound, but we would expect these mutations to affect binding affinity constants if the binding site were the same as in the CERK1 protein. Indeed, these substitutions led to the loss of ability to bind to PGN fragments, and drastically lowered affinities for chitin polymers as compared with the full-length protein (Table 3). To further complement the single LysM binding studies and to investigate the contribution of the individual LysM modules, we evaluated the effect of substitutions in LysM1 (Mut_LysM1) alone, and in
A
B
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Fig. 4. Homology modelling and docking of chitin in the CwlS LysM domains. (A) The upper panel shows the structure of the A. thaliana crystal structure of the CERK1 LysM2 domain bound to chitin [16]. The lower panel shows the homology modelling of the four LysM domains of CwlS. The dashed lines indicate the potential interaction (Van der Waals or hydrophobic) between the LysM residue and the N-acetyl group of the GlcNAc4 moiety of chitin. (B) Sequence alignment performed with CLUSTALX of LysM domains from different phyla and described in the literature. B.s., B. subtilis; M.o., Magnaporthe oryzae; C.f., Cladosporium fulvum; P.r., P. ryukyuensis; A.t., A. thaliana. The black arrows and red colour indicate the positions of the residues mutated in CwlS, and the cartoon below indicates the secondary structures as found in the CERK1 LysM2 domain crystal structure.
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Table 3. CwlS binding site mutant binding constants for chitin ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; >, saturation could not be reached.
FL-Mut FL-Mut_LysM1 FL-Mut_LysM1_2 FL-Mut_LysM1_3 FL-Mut_LysM1_4
Chitooctaose
Chitohexaose
Chitopentaose
Chitotetraose
> 698.75 1.64 13.84 8.03 2.52
> 1454.50 10.86 114.15 26.87 21.60
> 7293.00 176.05 51.05 > 2578.00 661.55 90.65 150.40 30.50
> 8304.50 328.60 64.60 > 3309.00 701.90 99.10 400.95 118.95
0.40 1.96 1.07 0.90
combination with one of the other LysM domains: LysM2 (Mut_LysM1_2), LysM3 (Mut_LysM1_3), and LysM4 (Mut_LysM1_4; Fig. 1). Mutation of LysM1 alone had no impact on the binding capacity, as the affinity for chitooctaose was 1.64 lM (Table 3), which was similar to the affinities of 4LysM (Table 2) and 3LysM (1.5 and 1.8 lM, respectively). Binding site substitutions in LysM1 in combination with LysM2 or LysM3, however, reduced binding by approximately one order of magnitude (13.8 and 8.0 lM, respectively). This is very similar to what we observed upon removal of two LysM domains. Finally, mutation of LysM4 together with LysM1 had no influence on the binding, further confirming that the fourth LysM domain has very poor binding capacity. Altogether, these data suggest that the binding site of the bacterial CwlS LysM domain is similar to that of CERK1 LysM2, as proposed by our homology modelling. The multiple LysM domains are flexible in solution The recent crystal structures of the plant CERK1 ectodomain and the Ecp6 protein, both possessing three LysM domains, showed a globular shape stabilized by disulfide bonds between the LysM modules. In bacteria, as there is no Cys residue allowing these bonds, the question of how the LysM domains are arranged in three dimensions is therefore still open. Despite numerous efforts, we could not crystallize CwlS. To gain insights into the arrangement of the four LysM domains, we performed solution structure analysis with small-angle X-ray scattering (SAXS). The data showed that the protein was not aggregated even at high concentrations (see Experimental procedures), and behaved as a monomer (Fig. 5). The q2 dependence (where q is the modulus of the scattering vector) at intermediate q indicated that CwlS is a very flexible protein and that it adopts an overall random-flight shape. The flexibility of the protein was further emphasized by modelling the data with the program CORAL (see Experimental procedures). The position, FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
1.67 27.75 7.29 5.38
orientation of the domains and conformation of the linkers were optimized against the scattering data in several independent optimizations. A representative result of the modelling from one optimization is presented in Fig. 5. The model is of very good quality, as it fits almost perfectly the experimental data, with a v-value of 1.65. The SAXS data and the modelling of CwlS show that the LysM modules do not adopt a globular shape in solution, and that the long linkers give flexibility to the LysM domains, allowing structural rearrangement. We further attempted to collect SAXS data on CwlS mixed with chitohexaose, to determine whether the LysM modules changed conformation upon sugar binding, but no structural rearrangement was observed (data not shown).
Discussion Our study shows, unequivocally, that bacterial LysM domains are involved in carbohydrate recognition, and that multiple LysM domain-containing proteins have a preference for long carbohydrate polymers. The number of LysM modules seems to be crucial for carbohydrate binding. In the CwlS endopeptidase, the four LysM domains have different binding capacities. LysM2 and LysM3 have, respectively, approximately twofold and threefold better binding affinities (Table 2) than LysM1, whereas LysM4 has only weak binding capacity. We infer that the weak LysM4 binding is not attributed to misfolding, as it behaves as a monomer in size exclusion chromatography. Furthermore, unfolded/aggregated proteins would be detected by MST, and this was not the case. Collectively, our observations regarding the endopeptidase activity, PGN pulldown assays and PGN and chitin polymer binding constants determined by MST demonstrate that at least two LysM domains are necessary for efficient binding to long chitin polymers, and that three LysM domains are necessary for proper binding to PGN fragments. These observations correlate with results showing that Enterococcus faecalis AtlA is much more active in the presence of its six LysM domains, and that AcmA autolysin from 1203
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Fig. 5. SAXS and modelling of the CwlS protein. The left panel shows the scattering curve of the full-length protein. The raw data are indicated by the dark squares. The black curve corresponds to the fit of the CORAL model to the SAXS data; the fit has a v-value of 1.65. The right model depicts a typical model obtained by modelling with CORAL. The LysM domains are shown in red, green, orange, and blue, and the catalytic domain and the linker are shown in black.
Lactococcus lactis needs at least two LysM domains for optimal activity in vivo [17,18]. Interestingly, most NlpC/P60 proteins found in the pFam database [19] have a minimum of two LysM domains. This conserved feature may indicate that proteins containing multiple LysM modules have been selected throughout evolution for optimal function. Binding experiments suggest different binding modes for short PGN fragments and chitin polymers. Single LysM domains (1LysM_1, 1LysM_2, 1LysM_3, or 1LysM_4) and 2LysM-Cata bind long chitin polymers, whereas binding of PGN fragments was not detected. This suggests that the degree of carbohydrate polymerization influences LysM domain cooperativity. Whereas the chitin polymers are linked together by glycosidic bonds, the GlcNAc–MurNAc units are connected only by the peptide crosslinks (Fig. 3). Long chitin polymers might allow multiple LysM domains to bind one carbohydrate polymer, enabling cooperativity between the LysM domains. The shorter PGN fragments that we used in this study may not allow two LysM domains to bind the same carbohydrate molecule, meaning that binding is not enhanced. However, our study does not reveal how cooperativity occurs, i.e. whether a carbohydrate chain bound to a specific LysM domain is, at the same time, able to engage another LysM domain of the same CwlS molecule or another LysM domain from a second CwlS molecule. As PGN is a very long polymer [20], we propose that such intramolecular and/or intermolecular cooperativity occurrs in vivo. The crystal structure of the Arabidopsis thaliana CERK1 ectodomain bound to chitin shows that one LysM domain can accommodate four sugar units. The same study proposed that two LysM domains can bind chitooctaose and trigger receptor dimerization [16]. It 1204
was proposed that the fungal Ecp6 protein containing three LysM domains behaves as a ‘three times one binding site’ when challenged with short chitin polymers in isothermal calorimetry. Nevertheless, cooperativity between LysM domains was suggested when chitooctaose was used as ligand [21]. Recent biochemical and structural data have shown that Ecp6 possesses two binding sites, one of high affinity in the picomolar range, and a second one in the micromolar range [22]. The crystal structure of A. thaliana CERK1 indicates, however, that only one of the three LysM domains is bound to chitin, and that the two other LysM domains play a structural role by maintaining the fold of the extracellular domain. Indeed, in plants and fungi, the multiple LysM domains are linked together by disulfide bonds. In contrast, these bonds are absent in bacteria, and the LysM modules are instead connected by linkers varying in length. Our SAXS data and modelling show that these linkers allow flexibility between the different LysM domains, and may therefore allow each of them to bind a carbohydrate molecule. Whereas our docking and mutagenesis studies show that the CwlS LysM binding site is similar to those of LysM proteins from other phyla, differences in binding strength are observed between LysM proteins from different phyla. Biochemical and/or in vivo binding studies of plant LysM proteins have shown affinities in the low nanomolar range for GlcNAc polymers [8,23–25]. Recently, the affinity for chitin polymers of fungal proteins was investigated. Ecp6 with three LysM repeats has low-picomolar and micromolar affinity for long chitin polymers [21,22]. Single LysM domains of plant and fungal chitinases have also micromolar affinity for chitin polymers [12,15]. These binding studies, FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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together with our binding analysis of bacterial LysM protein, demonstrate that affinities of LysM proteins for their cognate substrate range from low picomolar to high micromolar, and that LysM affinity cannot be predicted from phylogenetics. From our binding studies, we conclude that, in contrast to plant LysM domain-containing receptors, which have to discriminate between PGN, chitin polymers and lipo-oligosaccharide to distinguish between friends and foes [26–28], bacterial LysM domains might not have evolved to acquire high affinity for PGN, or to make distinctions among different GlcNAc polymers: first, because NlpC/P60 proteins are usually secreted in the vicinity of the cell wall, where PGN is highly concentrated, and therefore do not need to have high affinity for substrates; and second, because GlcNAc polymers, like chitin, are not produced in bacteria.
Experimental procedures Generation of plasmids The CwlS gene, excluding the sequence encoding the first 26 residues predicted to be the signal peptide (as determined by the SIGNALP 4.1 Server [29]), was amplified from B. subtilis genomic DNA with Phusion High-Fidelity DNA Polymerase (Finnzymes). The full-length protein, 4LysM and 1LysM_1 were cloned in-frame with the His-tag and S-tag into pET-30 Ek/LIC expression vectors, and the LysM-deleted versions – 3LysM-Cata, 2LysM-Cata, 1LysM-Cata, and Cata – were cloned in frame with the His-tag, S-tag and glutathione-S-transferase-tag into pET-41 Ek/LIC expression vectors (Novagen). 3LysM, 2LysM and 1LysM_4 were cloned in-frame with the His-tag, S-tag and thioredoxin-tag into pET-32 Ek/LIC expression vectors. The CwlS mutant genes, FL-Mut and derivatives were all cloned into the pGS-21a vector (containing both the His-tag and glutathione-S-transferase-tag), purchased from GenScript. A sequence encoding the tobacco etch virus protease cleavage site was introduced into all of the constructs to allow removal of the affinity tags during the protein purification process.
Protein expression and purification All of the protein derivatives were expressed and purified in the same manner. Proteins were expressed in E. coli BL21 CodonPlus RIL competent cells. An overnight culture grown in LB medium supplemented with antibiotics was diluted 100-fold in 12 L of the same medium, and incubated at 37 °C under agitation. When A600 nm reached 0.8, 1 mM isopropyl thio-b-D-galactoside was added to induce protein expression overnight at 20 °C with a rotational FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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speed of 150 rpm. The cells were pelleted by centrifugation at 12 000 g for 20 min, and resuspended in 100 mL of lysis buffer (50 mM Tris/HCl, pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol, 20 mM imidazole, 1 mM benzamidine). The cells were then lysed by sonication, and centrifuged at 30 000 g for 45 min to remove cell debris. All of the different proteins were purified with the same procedures, and all purification steps were performed at 4 °C. First, the crude extract was subjected to affinity chromatography with Ni2+–nitrilotriacetic acid superflow cartridges (Qiagen) pre-equilibrated with lysis buffer. The beads were then washed with high-salt buffer (50 mM Tris/HCl pH 8, 1 M NaCl, 5 mM b-mercaptoethanol, 50 mM imidazole, 1 mM benzamidine), and the proteins were eluted with elution buffer (50 mM Tris/HCl pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol, 500 mM imidazole, 1 mM benzamidine). Then, His-tagged tobacco etch virus protease was added to the eluted protein in a 1 : 50 (w/w) ratio, and the cleavage reaction was allowed to proceed overnight in a 12 000– 14 000-MWCO dialysis tube (6000–8000 MWCO for Cata; Spectrum Labs) against 1 L of dialysis buffer (50 mM Tris/ HCl pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol). For 2LysM protein purification, the NaCl concentration in the lysis, elution and dialysis buffer was 400 mM instead. After dialysis, the solutions were centrifuged at 3320 g for 30 min to remove aggregated proteins. A second round of nickel affinity chromatography was performed, and the cleaved protein was collected in the flow-through. Finally, the proteins were concentrated to 2 mgmL 1 with 10 000MWCO Vivaspin centrifugal concentrators (Sartorius Stedim, Goettingen, Germany), and further purified by size exclusion chromatography with a Superdex75 10/300 GL column (GE Healthcare, Brondy, Denmark) connected to € an Akta purifier system (GE Healthcare). The proteins were eluted with gel filtration buffer (GFB; 50 mM Tris/ HCl, pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol). All recombinant proteins were at least 95% pure after these purification steps (Fig. 2B).
Carbohydrate polymers Soluble fragments of bacterial cell wall were purchased from CeCo Labs (http://www.cecolabs.de). The S. aureus and B. subtilis cell walls were treated with glucosaminidase; therefore, the purified fragments contained only GlcNAc– MurNAc disaccharides linked by the peptide stem (Fig. 3). The PGN fragments had high purity and were free of lipopolysaccharides. Chitin oligomers were purchased from Seikagaku Biobusiness (Tokyo, Japan) and Sigma-Aldrich (Copenhagen, Denmark).
MST Protein interactions with bacterial cell wall fragments (CeCo Labs) and chitin oligomers (Seikagaku Biobusiness and
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Sigma-Aldrich) were measured with MST. Proteins were labelled with the Monolith NT.115 Protein Labelling Kit BLUE (NanoTemper Technologies, Munich, Germany) to achieve a 1 : 1 molar ratio of labelled protein to dye. A titration series was prepared, in which the concentration of the labelled proteins was kept constant at 100 nM, and the concentration of the titrant (octa-N-acetyl-chitooctaose, hexa-N-acetyl-chitohexaose, penta-N-acetyl-chitopentaose, tetra-N-acetyl-chitotetraose, S. aureus tetramer, S. aureus trimer, B. subtilis trimer, or B. subtilis dimer) was varied in thermophoresis buffer (50 mM phosphate, pH 7.5, 1 M NaCl, 0.1% Tween-20). After an incubation period of 20 min, MST measurements were performed at 25 °C on a Monolith NT.115 instrument (NanoTemper Technologies). For each measurement, the laser was switched on for 30 s and off for 5 s. Binding curves were obtained from the thermophoresis plus jump phase, with IR laser power of 20% or 50%. The negative control for each protein was performed with 100 nM labelled protein in thermophoresis buffer in all 16 capillaries under the same conditions mentioned above. Each experiment was repeated at least three times, and an average KD value was calculated after analysis of the data with GRAPHPAD PRISM.
Endopeptidase activity assay An overnight B. subtilis cell culture (ATCC 6056) was grown in 100 mL of LB medium at 30 °C, with a rotational speed of 120 rpm. One millilitre of the overnight culture was grown in fresh LB medium under similar conditions. This procedure was repeated by diluting the overnight culture five times in fresh LB medium, and the cells were used for turbidity assessment when a A600 nm reading of 1.2–1.3 was achieved. All attenuance readings for the assay were obtained with a Jenway 6300 spectrophotometer. The B. subtilis cells in the mid-exponential to late exponential growth phase were diluted with LB medium to obtain a A600 nm reading of ~ 0.9. Each protein (40 lM) in GFB was added to the diluted cells at a 1 : 1 (v/v) ratio, and the mixture was incubated in a cuvette at 30 °C, with a rotational speed of 120 rpm. A A600 nm reading was recorded at regular intervals of 20 min over a period of 140 min. The positive and negative controls were performed by adding 120 lM lysozyme in GFB, or GFB alone, to the diluted cells. The specific activity reflecting three independent experiments corresponds to the loss of A600 nm, and is expressed in lDmin 1lg 1.
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1 M NaCl, 5 mM b-mercaptoethanol, 1% Tween-20). The reaction mixture was incubated for 20 min at 22 °C, and centrifuged at 10 500 g for 45 min. Then, the supernatant was collected, and the insoluble PGN pellet was resuspended in 15 lL of assay buffer. Ten microlitres of supernatant and the resuspended pellet solution were analysed on an 18% SDS/PAGE gel. The results from five experiments were processed with TOTALLAB QUANT software (BioStep GmbH, Jahnsdorf, Germany). The protein densities from the supernatant and pellet were summed, and from these the proportions of protein densities in the supernatant and pellet (averaged over all experiments) were derived and compared. A negative control was performed each time with 10 lg of each protein in the same conditions as mentioned above, without the PGN, to ensure that the proteins were not precipitating on their own. The binding capacity is expressed as percentages of protein present in the pellet and supernatant.
Homology modelling Homology modelling was performed with the automated mode of the Swiss-model server [30]. Several improvements were made to the models, notably by manually adjusting the Ramachandran plot and rotamer residues with COOT [31].
SAXS SAXS data were recorded at 25 °C and at a wavelength of on a prototype of the NanoSTAR camera from Bru1.54 A ker AXS that has been optimized for solution scattering [32]. The acquisition time depended on concentration. Initial data treatment, buffer subtraction and conversion to absolute scale, with water as a standard, were performed with the SUPERSAXS program package (Cristiano Luis Pinto de Oliveira, Institute of Physics, University of Sa˜o Paulo, Sa˜o Paulo, Brazil and J.S.P., unpublished). The intensity is presented as a function of the scattering vector q = 4p sin(h)/k, where k is the wavelength and 2h is the scattering angle. The forward scattering used to determine the molecular mass of the particles in solution was determined with the indirect Fourier transform procedure [33] implemented in the home-written program WIFT [34,35]. Measurements were performed at two different protein concentrations: 4.7 and 1.7 mgmL 1. Rigid body modelling was performed with CORAL [36], with the homology models of the catalytic and LysM domains generated as described above as input.
Insoluble PGN pulldown assay PGN from B. subtilis (Sigma-Aldrich) was dissolved in Mops/NaOH (pH 7) to a final concentration of 10 mgmL 1. Prior to the assay, PGN was heated at 100 °C for 10 min to denature remnant proteases. In a 50lL reaction mixture, 10 lg of each rotein was added to 50 lg of PGN in the assay buffer (50 mM Tris/HCl, pH 8, 1206
Acknowledgements This research was funded by Danish National Research Foundation, grant no. DNRF79. We thank F. Dolman and D. Reid for critical review of the manuscript, and J. Bauer for fruitful discussions. FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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References 1 Typas A, Banzhaf M, Gross CA & Vollmer W (2011) From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10, 123–136. 2 Anantharaman V & Aravind L (2003) Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes. Genome Biol 4, R11. 3 Xu Q, Sudek S, McMullan D, Miller MD, Geierstanger B, Jones DH, Krishna SS, Spraggon G, Bursalay B, Abdubek P et al. (2009) Structural basis of murein peptide specificity of a gamma-D-glutamyl-l-diamino acid endopeptidase. Structure 17, 303–313. 4 Buist G, Steen A, Kok J & Kuipers OP (2008) LysM, a widely distributed protein motif for binding to (peptido) glycans. Mol Microbiol 68, 838–847. 5 Petrovic DM, Leenhouts K, van Roosmalen ML, Kleinjan F & Broos J (2012) Monitoring lysin motif– ligand interactions via tryptophan analog fluorescence spectroscopy. Anal Biochem 428, 111–118. 6 Fukushima T, Afkham A, Kurosawa S, Tanabe T, Yamamoto H & Sekiguchi J (2006) A new D, L-endopeptidase gene product, YojL (renamed CwlS), plays a role in cell separation with LytE and LytF in Bacillus subtilis. J Bacteriol 188, 5541–5550. 7 Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A et al. (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301–315. 8 Broghammer A, Krusell L, Blaise M, Sauer J, Sullivan JT, Maolanon N, Vinther M, Lorentzen A, Madsen EB, Jensen KJ et al. (2012) Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc Natl Acad Sci USA 109, 13859–13864. 9 Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N et al. (2003) Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425, 585–592. 10 Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H & Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 104, 19613–19618. 11 Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D, Tsuda K, Katagiri F, Fliegmann J, Bono JJ, Cullimore JV et al. (2011) Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci USA 108, 19824–19829.
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12 Ohnuma T, Onaga S, Murata K, Taira T & Katoh E (2008) LysM domains from Pteris ryukyuensis chitinaseA: a stability study and characterization of the chitinbinding site. J Biol Chem 283, 5178–5187. 13 Rouge P, Nerinckx W, Gough C, Bono JJ & Barre A (2011) Docking of chitin oligomers and Nod factors on lectin domains of the LysM-RLK receptors in the Medicago–Rhizobium symbiosis. Adv Exp Med Biol 705, 511–521. 14 Radutoiu S, Madsen LH, Madsen EB, Jurkiewicz A, Fukai E, Quistgaard EM, Albrektsen AS, James EK, Thirup S & Stougaard J (2007) LysM domains mediate lipochitin–oligosaccharide recognition and Nfr genes extend the symbiotic host range. EMBO J 26, 3923–3935. 15 Koharudin LM, Viscomi AR, Montanini B, Kershaw MJ, Talbot NJ, Ottonello S & Gronenborn AM (2011) Structure–function analysis of a CVNH-LysM lectin expressed during plant infection by the rice blast fungus Magnaporthe oryzae. Structure 19, 662–674. 16 Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J et al. (2012) Chitin-induced dimerization activates a plant immune receptor. Science 336, 1160–1164. 17 Eckert C, Lecerf M, Dubost L, Arthur M & Mesnage S (2006) Functional analysis of AtlA, the major N-acetylglucosaminidase of Enterococcus faecalis. J Bacteriol 88, 8513–8519. 18 Steen A, Buist G, Horsburgh GJ, Venema G, Kuipers OP, Foster SJ & Kok J (2005) AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning. FEBS J 272, 2854–2868. 19 Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J et al. (2012) The Pfam protein families database. Nucleic Acids Res 40, 290–301. 20 Hayhurst EJ, Kailas L, Hobbs JK & Foster SJ (2008) Cell wall peptidoglycan architecture in Bacillus subtilis. Proc Natl Acad Sci USA 105, 14603–14608. 21 de Jonge R, van Esse HP, Kombrink A, Shinya T, Desaki Y, Bours R, van der Krol S, Shibuya N, Joosten MH & Thomma BP (2010) Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science 329, 953–955. 22 Sanchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg DJ, Thomma BP & Mesters JR (2013) Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. Elife 2, e00790. 23 Shimizu T, Nakano T, Takamizawa D, Desaki Y, IshiiMinami N, Nishizawa Y, Minami E, Okada K, Yamane H, Kaku H et al. (2010) Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice. Plant J 64, 204–214.
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24 Iizasa E, Mitsutomi M & Nagano Y (2010) Direct binding of a plant LysM receptor-like kinase, LysM RLK1/CERK1, to chitin in vitro. J Biol Chem 285, 2996–3004. 25 Petutschnig EK, Jones AM, Serazetdinova L, Lipka U & Lipka V (2010) The lysin motif receptor-like kinase (LysM-RLK) CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation. J Biol Chem 285, 28902–28911. 26 Oldroyd GE (2013) Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11, 252–263. 27 Madsen LH, Tirichine L, Jurkiewicz A, Sullivan JT, Heckmann AB, Bek AS, Ronson CW, James EK & Stougaard J (2010) The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus. Nat Commun 1, 1–12. 28 Gust AA, Willmann R, Desaki Y, Grabherr HM & N€ urnberger T (2012) Plant LysM proteins: modules mediating symbiosis and immunity. Trends Plant Sci 17, 495–502. 29 Petersen TN, Brunak S, von Heijne G & Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8, 785–786.
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30 Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J & Schwede T (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4, 1–13. 31 Emsley P, Lohkamp B, Scott WG & Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501. 32 Pedersen JS (2004) A flux- and background-optimized version of the NanoSTAR small-angle X-ray scattering camera for solution scattering. J Appl Crystallogr 37, 369–380. 33 Glatter O (1977) A new method for the evaluation of small-angle scattering data. J Appl Crystallogr 10, 415–421. 34 Oliveira CL, Behrens MA, Pedersen JS, Erlacher K, Otzen D & Pedersen JS (2009) A SAXS study of glucagon fibrillation. J Mol Biol 387, 147–161. 35 Pedersen JS, Hansen S & Bauer R (1994) The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering. Eur Biophys J 22, 379–389. 36 Petoukhov MV, Franke D, Shkumatov AV, Tria G, Kikhney AG, Gajda M, Gorba C, Mertens HDT, Konarev PV & Svergun DI (2012) New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr 45, 342–350.
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Keywords autolysin; chitin; lysin motif (LysM) domain; NlpC/P60 endopeptidase; peptidoglycan Correspondence M. Blaise, Department of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling, Aarhus University, Gustav Wieds Vej 10, Aarhus 8000, Denmark Fax: +45 8612 3178 Tel: +45 8715 5524 E-mail: [email protected] *Present address: Statens Serum Institut, Copenhagen University, Artillerivej 5, Copenhagen S, 2300, Denmark (Received 30 June 2013, revised 12 December 2013, accepted 17 December 2013) doi:10.1111/febs.12698
Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these b,1–4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydratebinding site in LysM-containing proteins is conserved across phyla.
Introduction The bacterial cell wall consists of a rigid layer of peptidoglycan (PGN) that maintains the integrity of the bacterial cell and confers protection against environmental stress. PGN is a polymer of GlcNAc and N-acetylmuramic acid (MurNAc) arranged in a three-dimensional network crosslinked by peptide chains that connect MurNAc moieties. Although PGN is rigid, it also has to be highly dynamic to allow the bacterial cells to elongate and separate during growth and division [1]. Numerous enzymes grouped under the
family of autolysins are involved in PGN degradation and remodelling, thereby contributing significantly to PGN structural dynamics. These enzymes have diverse activities: glycosidases are lysozyme-type enzymes that cleave the carbohydrate chain of PGN, whereas endopeptidases, amidases and carboxypeptidases hydrolyse the peptide stem of PGN at different positions. Among the autolysins, NlpC/P60 D,L-endopeptidases play a crucial role in the remodelling of PGN. Most D, L-endopeptidases cleave the peptide chain of PGN
Abbreviations CwlS, cell wall-lytic enzyme associated with cell separation; GFB, gel filtration buffer; GlcNAc, N-acetylglucosamine; LysM, lysine motif; MST, microscale thermophoresis; MurNAc, N-acetylmuramic acid; PGN, peptidoglycan; SA, specific activity; SAXS, small-angle X-ray scattering.
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between the second and third residues. In bacteria, NlpC/P60 enzymes are modular proteins composed of a catalytic domain, named NlpC/P60, that is very often associated with an additional domain(s) predicted to be involved in PGN binding. Indeed, the SH3b, cholinebinding domain and lysin motif (LysM) [2,3] domains are very often found in NlpC/P60 endopeptidases. The LysM domain, a small protein domain of ~ 50 residues, has been defined as a carbohydrate-binding domain with affinity for GlcNac polymers [4]. In bacteria, the LysM domain is often associated with proteins involved in PGN synthesis or remodelling, and several LysM domains are frequently found in these enzymes [4]. Few biochemical studies have demonstrated direct binding between LysM domains and PGN, and the binding studies have typically been conducted with insoluble PGN for affinity pulldown assays. Rigorous determination of the substrate affinity of bacterial LysM domains has not been achieved, as biochemical studies are hampered by the properties of PGN. PGN is insoluble when extracted from bacteria, and PGN oligomers are difficult to synthesize or purify in large amounts. Thus, it is not trivial to perform PGN-binding experiments to assess binding affinities. Illustrating this complexity, binding of the LysM-containing protein Lactococcus lactis AcmA to bacterium-like particles and PGN was shown by steady-state fluorescence, but it was not possible to determine affinity constants [5]. In summary, despite recent progress in studying bacterial LysM-containing proteins, several questions
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remain unanswered. Notably, these concern the affinities for their ligands, the correlation between the number of LysM modules and the affinity for carbohydrates, the ligand recognition specificity, and, finally, the binding site localization. In this study, we addressed these questions by using biochemical approaches to study cell wall-lytic enzyme associated with cell separation (CwlS), which is involved in cell separation during division of Bacillus subtilis [6]. CwlS is an NlpC/P60 D,L-endopeptidase, possessing four LysM domains in the N-terminus followed by a C-terminal NlpC/P60 catalytic domain [2]. It is therefore an appropriate protein model for investigating the individual and cooperative role(s) of LysM domains in carbohydrate binding.
Results Expression and purification of CwlS To investigate the influence of LysM modules on the binding affinity of CwlS, we first purified the CwlS fulllength protein (FL) and CwlS catalytic domain (Cata), by using the Escherichia coli expression system. Subsequently, we expressed and purified a series of protein derivatives (Fig. 1). The N-terminally to C-terminally LysM-truncated mutants lack one (3LysM-Cata), two (2LysM-Cata) or three (1LysM-Cata) of the LysM domains in the N-terminus; the C-terminally to N-terminally LysM-truncated proteins mutants lack the catalytic domain (4LysM) plus one (3LysM), or two
Fig. 1. Schematic of the constructs used in this study. The LysM domains are shown in grey, and the catalytic domain in light grey. The residue numbering is indicated. The mutations of the LysM domain are indicated by ‘A’ followed by a number giving the position.
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(2LysM) LysM domains in the C-terminus. To examine the binding capacity of individual LysM domains, single LysM domains (1LysM_1, 1LysM_2, 1LysM_3 and 1LysM_4) numbered 1–4 from the N-terminus were also expressed and purified. Finally, to assess the carbohydrate recognition mode and to further supplement the single LysM studies, we used an Ala scanning mutagenesis approach to express and purify a series of binding site variants (FL_Mut and FL_Mut1, FL_Mut1_2, FL_Mut1_3, and FL_Mut1_4). All proteins could be purified to ~ 95% purity and appeared as a single homogeneous peak in size exclusion chromatography, strongly indicating that they are monomeric and folded. The number of LysM modules affects the endopeptidase activity of CwlS We first wanted to determine whether deletion of LysM domains might influence the endopeptidase activity and thereby the cell wall-degrading capacity of CwlS. For this purpose, purified proteins were added to exponentially growing B. subtilis cultures, and cell densities were measured. Addition of purified full-length protein resulted in decreased cell density over time with a specific activity (SA) of 99 U (where U is defined as lAmin 1lg 1 of enzyme; see Experimental procedures), demonstrating that the purified protein was active (Fig. 2A). In this assay, 3LysM-Cata seemed to be less active, with an SA of 68 U. The loss of activity was even more pronounced when two or three LysM domains were deleted (2LysM-Cata, SA = 33 U; 1LysM-Cata, SA = 28 U). Moreover, the cell density did not decrease over time when all LysM domains were removed, suggesting an absence of endopeptidase activity on bacterial cell walls. The number of LysM modules affects PGN binding To assess whether the correlation of the decreased endopeptidase activity and decreased number of LysM modules was linked to the loss of PGN-binding capacity, we performed semiquantitative PGN pulldown assays (Fig. 2B,C). This assay exploits the insoluble nature of bulk PGN. Proteins binding to insoluble PGN will therefore coprecipitate with PGN and be found in the pellet after centrifugation. The PGN pulldown experiments showed that the full-length protein and 3LysM-Cata had similar binding capacities, as approximately 44% and 45% of the proteins were pelleted (Fig. 2C). However, binding strength appeared to decrease with the number of LysM modules present, as only 28%, 20% and 18% 1198
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of 2LysM-Cata, 1LysM-Cata, and the catalytic domain, respectively, bound to PGN (Fig. 2C). This suggests that the binding affinity correlates with the number of LysM modules, and that the catalytic domain has only low affinity for PGN. LysM domains bind PGN fragments in the lowmicromolar range To further characterize the affinity for PGN, we assessed the binding of CwlS proteins to different polymer lengths of commercially available PGN fragments (Fig. 3) purified from Staphylococcus aureus or B. subtilis. In this assay, microscale thermophoresis (MST) [7], a rather new technique that has been proven to be adequate for binding studies with aqueousphase carbohydrates, was used [8]. We first tested a soluble PGN tetramer from S. aureus and a soluble PGN trimer from B. subtilis (Fig. 3). Both were extracted from cell walls treated with glucosaminidase and endopeptidase, which leaves only GlcNAc–MurNAc disaccharides. The carbohydrate polymerization is maintained by the peptide chain, as the MurNAc– GlcNAc bond has been hydrolysed (Fig. 3). MST experiments showed that the full-length protein had an equilibrium dissociation constant (KD) of 10.1 lM for S. aureus PGN tetramer (Table 1). Interaction also occurred when shorter PGN fragments were measured in this assay, but the binding affinities of S. aureus PGN trimer and B. subtilis PGN trimer were approximately one order of magnitude lower than that of S. aureus PGN tetramer (Table 1). In addition, the affinity for B. subtilis PGN dimer was ~ 30-fold lower than that for S. aureus PGN tetramer (Table 1). This demonstrates that bacterial LysM domains have an affinity for PGN fragments in the low-macromolar range. Furthermore, our data suggest that LysM domains have higher affinity for longer carbohydrate polymers. LysM multiplicity increases affinity for PGN To investigate the influence of LysM modules on binding affinity, we utilized the protein set where LysM domains had been removed one by one from the N-terminus to the C-terminus (Fig. 1). The binding capacities of these proteins were tested in aqueous phase with MST. 3LysM-Cata had a similar affinity for S. aureus PGN tetramer as the full-length protein, considering that the KD value was ~ 9 lM. However, removal of two or three LysM domains influenced the binding to PGN. Indeed, binding to PGN was not detectable for either 2LysM-Cata, 1LysM-Cata, 1LysM_1, or 1LysM_4. In contrast, a protein derivative possessing FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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120
A
U (μD/min/μg)
100 80 60 40 20 0 FL
B FL kDa
P
SN
3LysM 2LysM 1LysM Cata Cata Cata
Control 3LysM 2LysM 1LysM Cata Cata Cata P SN P SN P SN
Cata
Lysozyme Buffer
Cata P
SN
kDa
116 66 45
40
35
25
25 18
15 14 10
Fig. 2. (A) Endopeptidase activity assay. The graph shows the D600 nm of B. subtilis cell cultures after protein addition. The protein set used in this assay is described in Fig. 1. Lysozyme and buffer were used as positive and negative controls, respectively. (B, C) Semiquantitative pulldown experiments. (B) Control experiment in which no PGN was added to the protein, to verify that proteins were not precipitating on their own after centrifugation. (C) Gel and quantification table of the pulldown experiment results. P, pellet; SN, supernatant. The table represents an average of four independent pulldown experiments.
C FL kDa
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only the four LysM domains (4LysM) had similar affinity as the full-length protein, as it binds S. aureus PGN tetramer with an affinity of 6.8 lM. This shows that the binding contribution of the catalytic domain is negligible. This conclusion is reinforced by the fact that binding of the catalytic domain alone to PGN fragments was not detectable (Table 1). Our experiments clearly indicate that LysM domains are crucial for protein binding to PGN, and that the contribution of the catalytic domain to substrate recognition is minor. FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
+ PGN 2LysM Cata P SN
10
CwlS protein
Proportion in pellet
FL
0.44 ± 0.08
3LysM-Cata
0.45 ± 0.11
2LysM-Cata
0.28 ± 0.05
1LysM-Cata
0.20 ± 0.08
Cata
0.17 ± 0.08
CwlS LysM domains do not discriminate between GlcNAc polymers and MurNAc–GlcNAc polymers In plants, a variety of membrane receptors containing LysM domains are involved in recognition of GlcNAc polymers. Although these receptors are very similar to each other, they distinguish between three related substrates – lipo-chitooligosaccharide, chitin polymers, or PGN – whose recognition must be specific, given that each of these carbohydrate polymers triggers a different
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HO HO HO
HO
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HO
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HO NH
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S. aureus PGN tetramer HO HO HO
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l-Ala l-Ala
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B. subtilis PGN trimer
B. subtilis PGN dimer
Fig. 3. Structure of the PGN fragments used in this study. The arrows indicate that the MurNAc–GlcNac glycosidic bond is not preserved in these ligands. mA2 pm, meso-diaminopimelic acid.
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Table 1. CwlS binding constants for PGN ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; NT, not tested.
Full-length protein 3LysM-Cata 2LysM-Cata 1LysM-Cata Catalytic domain 4LysM FL-Mut
Staphylococcus aureus tetramer
Staphylococcus aureus trimer
Bacillus subtilis trimer
Bacillus subtilis dimer
10.11 2.08 9.07 1.73 ND ND ND 6.81 1.31 ND
173.20 59.80 170.40 33.50 NT NT NT 479.90 211.30 NT
121.02 40.18 187.30 73.00 ND NT ND 207.80 72.70 NT
356.90 98.10 134.40 20.60 NT NT NT 222.45 56.85 NT
plant response pathway [9–11]. They lead either to plant defence (for chitin and PGN) or to symbiosis (lipo-chitooligosaccharide). Following this line of reasoning, we tested whether a bacterial LysM protein could discriminate between PGN and chitin GlcNAc polymers. Our MST binding experiments showed that the fulllength protein had a KD of 4.7 lM for chitooctaose (Table 2). As seen with the PGN fragments, the affinity decreased when shorter chitin polymers were assayed; chitohexaose, chitopentaose and chitotetraose, respectively, had KD values of 20.4, 316.4 and 765.7 lM. To extensively investigate the binding properties of a multiple LysM bacterial protein, we assessed the chitin-binding capacities of all of the CwlS protein derivatives (Fig. 1). The affinity was similar for chitooctaose when the full-length protein, 3LysM-Cata and 2LysMCata were compared, but only weak binding was observed for 1LysM-Cata (Table 2). Interestingly, 1LysM_1, which did not bind PGN fragments, was shown to bind chitin polymers with a KD of 21.2 lM, which is similar to value for the single LysM domain from Pteris ryukyuensis chitinase-A [12]. We further tested the other LysM domains to assess their individual binding affinities. 1LysM_2 and 1LysM_3 had higher affinity for chitin oligomers than 1LysM_1 (9.6
and 7.6 versus 21.3 lM, respectively, for chitooctaose), whereas only weak binding of chitooctaose was detected with 1LysM_4. This indicates that the different LysM domains have different binding affinities. To further explore this hypothesis, the chitin-binding capacities of 3LysM (LysM1 to LysM3) and 2LysM (LysM1 to LysM2) were assessed. These proteins had affinities similar to those of 3LysM-Cata and 2LysMCata (Table 2). Together, these data indicate that LysM1, LysM2 and LysM3 have similar affinities, whereas LysM4 seems to bind carbohydrates only weakly, as observed in the PGN pulldown assay (Fig. 2C) and MST assay (Table 2). Congruent with the MST results observed for PGN fragments, the binding affinities of the single LysM domains gradually decreased with a reduction in the length of the chitin polymers (Table 2). Similarity between the CwlS LysM carbohydratebinding sites and other LysM domains Several groups have proposed different binding sites for the LysM domains from different phyla, on the basis of docking and molecular dynamics [13,14]. Likewise, NMR studies have suggested residues that might
Table 2. CwlS binding constants for chitin ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; NT, not tested; >, saturation could not be reached.
Full-length protein 3LysM-Cata 2LysM-Cata 1LysM-Cata Catalytic domain 4LysM 3LysM 2LysM 1LysM_1 1LysM_2 1LysM_3 1LysM_4
Chitooctaose
Chitohexaose
Chitopentaose
Chitotetraose
4.78 0.58 3.13 0.77 9.89 2.19 > 1089.40 ND 1.53 0.07 1.83 0.17 7.88 1.11 21.29 1.16 9.63 1.41 7.61 1.80 > 1312.55
20.40 11.63 18.06 ND ND 14.15 12.74 24.34 44.74 12.17 9.92 ND
316.45 275.60 563.70 ND ND 288.10 243.04 393.75 520.40 328.75 186.45 NT
765.75 582.65 ND ND ND 810.65 444.95 483.05 787.85 384.20 > 1410.55 NT
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2.31 3.88 2.95
1.08 1.26 2.12 5.60 1.84 3.01
65.15 52.50 119.50
18.70 30.25 61.75 66.30 61.05 41.45
123.75 146.55
74.05 51.45 27.25 69.95 83.50
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be involved [12,15]. More recently, the crystal structure of the Arabidopsis chitin receptor CERK1 ectodomain–chitin complex has shed light on the binding site of a plant LysM2 domain. The CERK1 structure shows that chitin is bound by hydrophobic, Van der Waals and hydrogen bonds. At the reducing end of the carbohydrate, three residues, Met127, Gln131, and Ala138, recognized the N-acetyl group of GlcNAc4 (Fig. 4) [16]. To determine whether there are any similarities between the CERK1 LysM2 and bacterial LysM binding sites, we made homology models of the four LysM domains of CwlS and superposed them on the LysM2 domain of the CERK1 crystal structure. These models predict that Ile48, Lys52 and Thr60 from LysM1, Val109, Lys113 and Asp121 from LysM2, Val178, Lys182 and Asp190 from LysM3 and Val246, Arg250 and Asp258 from LysM4 could possibly engage in
J. E. M. M. Wong et al.
hydrophobic and Van der Waals interactions with GlcNAc4 N-acetyl group (Fig. 4). To test this hypothesis, we replaced these three residues with Ala in the four LysM domains, and determined the binding capacity of this protein derivative. These mutations are not expected to totally abolish the binding, because, in the CERK1 crystal structure, GlucNac residues 1–3 are also tightly bound, but we would expect these mutations to affect binding affinity constants if the binding site were the same as in the CERK1 protein. Indeed, these substitutions led to the loss of ability to bind to PGN fragments, and drastically lowered affinities for chitin polymers as compared with the full-length protein (Table 3). To further complement the single LysM binding studies and to investigate the contribution of the individual LysM modules, we evaluated the effect of substitutions in LysM1 (Mut_LysM1) alone, and in
A
B
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Fig. 4. Homology modelling and docking of chitin in the CwlS LysM domains. (A) The upper panel shows the structure of the A. thaliana crystal structure of the CERK1 LysM2 domain bound to chitin [16]. The lower panel shows the homology modelling of the four LysM domains of CwlS. The dashed lines indicate the potential interaction (Van der Waals or hydrophobic) between the LysM residue and the N-acetyl group of the GlcNAc4 moiety of chitin. (B) Sequence alignment performed with CLUSTALX of LysM domains from different phyla and described in the literature. B.s., B. subtilis; M.o., Magnaporthe oryzae; C.f., Cladosporium fulvum; P.r., P. ryukyuensis; A.t., A. thaliana. The black arrows and red colour indicate the positions of the residues mutated in CwlS, and the cartoon below indicates the secondary structures as found in the CERK1 LysM2 domain crystal structure.
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Table 3. CwlS binding site mutant binding constants for chitin ligands. The equilibrium dissociation constants (KD) are given in lM. ND, not detectable; >, saturation could not be reached.
FL-Mut FL-Mut_LysM1 FL-Mut_LysM1_2 FL-Mut_LysM1_3 FL-Mut_LysM1_4
Chitooctaose
Chitohexaose
Chitopentaose
Chitotetraose
> 698.75 1.64 13.84 8.03 2.52
> 1454.50 10.86 114.15 26.87 21.60
> 7293.00 176.05 51.05 > 2578.00 661.55 90.65 150.40 30.50
> 8304.50 328.60 64.60 > 3309.00 701.90 99.10 400.95 118.95
0.40 1.96 1.07 0.90
combination with one of the other LysM domains: LysM2 (Mut_LysM1_2), LysM3 (Mut_LysM1_3), and LysM4 (Mut_LysM1_4; Fig. 1). Mutation of LysM1 alone had no impact on the binding capacity, as the affinity for chitooctaose was 1.64 lM (Table 3), which was similar to the affinities of 4LysM (Table 2) and 3LysM (1.5 and 1.8 lM, respectively). Binding site substitutions in LysM1 in combination with LysM2 or LysM3, however, reduced binding by approximately one order of magnitude (13.8 and 8.0 lM, respectively). This is very similar to what we observed upon removal of two LysM domains. Finally, mutation of LysM4 together with LysM1 had no influence on the binding, further confirming that the fourth LysM domain has very poor binding capacity. Altogether, these data suggest that the binding site of the bacterial CwlS LysM domain is similar to that of CERK1 LysM2, as proposed by our homology modelling. The multiple LysM domains are flexible in solution The recent crystal structures of the plant CERK1 ectodomain and the Ecp6 protein, both possessing three LysM domains, showed a globular shape stabilized by disulfide bonds between the LysM modules. In bacteria, as there is no Cys residue allowing these bonds, the question of how the LysM domains are arranged in three dimensions is therefore still open. Despite numerous efforts, we could not crystallize CwlS. To gain insights into the arrangement of the four LysM domains, we performed solution structure analysis with small-angle X-ray scattering (SAXS). The data showed that the protein was not aggregated even at high concentrations (see Experimental procedures), and behaved as a monomer (Fig. 5). The q2 dependence (where q is the modulus of the scattering vector) at intermediate q indicated that CwlS is a very flexible protein and that it adopts an overall random-flight shape. The flexibility of the protein was further emphasized by modelling the data with the program CORAL (see Experimental procedures). The position, FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
1.67 27.75 7.29 5.38
orientation of the domains and conformation of the linkers were optimized against the scattering data in several independent optimizations. A representative result of the modelling from one optimization is presented in Fig. 5. The model is of very good quality, as it fits almost perfectly the experimental data, with a v-value of 1.65. The SAXS data and the modelling of CwlS show that the LysM modules do not adopt a globular shape in solution, and that the long linkers give flexibility to the LysM domains, allowing structural rearrangement. We further attempted to collect SAXS data on CwlS mixed with chitohexaose, to determine whether the LysM modules changed conformation upon sugar binding, but no structural rearrangement was observed (data not shown).
Discussion Our study shows, unequivocally, that bacterial LysM domains are involved in carbohydrate recognition, and that multiple LysM domain-containing proteins have a preference for long carbohydrate polymers. The number of LysM modules seems to be crucial for carbohydrate binding. In the CwlS endopeptidase, the four LysM domains have different binding capacities. LysM2 and LysM3 have, respectively, approximately twofold and threefold better binding affinities (Table 2) than LysM1, whereas LysM4 has only weak binding capacity. We infer that the weak LysM4 binding is not attributed to misfolding, as it behaves as a monomer in size exclusion chromatography. Furthermore, unfolded/aggregated proteins would be detected by MST, and this was not the case. Collectively, our observations regarding the endopeptidase activity, PGN pulldown assays and PGN and chitin polymer binding constants determined by MST demonstrate that at least two LysM domains are necessary for efficient binding to long chitin polymers, and that three LysM domains are necessary for proper binding to PGN fragments. These observations correlate with results showing that Enterococcus faecalis AtlA is much more active in the presence of its six LysM domains, and that AcmA autolysin from 1203
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Fig. 5. SAXS and modelling of the CwlS protein. The left panel shows the scattering curve of the full-length protein. The raw data are indicated by the dark squares. The black curve corresponds to the fit of the CORAL model to the SAXS data; the fit has a v-value of 1.65. The right model depicts a typical model obtained by modelling with CORAL. The LysM domains are shown in red, green, orange, and blue, and the catalytic domain and the linker are shown in black.
Lactococcus lactis needs at least two LysM domains for optimal activity in vivo [17,18]. Interestingly, most NlpC/P60 proteins found in the pFam database [19] have a minimum of two LysM domains. This conserved feature may indicate that proteins containing multiple LysM modules have been selected throughout evolution for optimal function. Binding experiments suggest different binding modes for short PGN fragments and chitin polymers. Single LysM domains (1LysM_1, 1LysM_2, 1LysM_3, or 1LysM_4) and 2LysM-Cata bind long chitin polymers, whereas binding of PGN fragments was not detected. This suggests that the degree of carbohydrate polymerization influences LysM domain cooperativity. Whereas the chitin polymers are linked together by glycosidic bonds, the GlcNAc–MurNAc units are connected only by the peptide crosslinks (Fig. 3). Long chitin polymers might allow multiple LysM domains to bind one carbohydrate polymer, enabling cooperativity between the LysM domains. The shorter PGN fragments that we used in this study may not allow two LysM domains to bind the same carbohydrate molecule, meaning that binding is not enhanced. However, our study does not reveal how cooperativity occurs, i.e. whether a carbohydrate chain bound to a specific LysM domain is, at the same time, able to engage another LysM domain of the same CwlS molecule or another LysM domain from a second CwlS molecule. As PGN is a very long polymer [20], we propose that such intramolecular and/or intermolecular cooperativity occurrs in vivo. The crystal structure of the Arabidopsis thaliana CERK1 ectodomain bound to chitin shows that one LysM domain can accommodate four sugar units. The same study proposed that two LysM domains can bind chitooctaose and trigger receptor dimerization [16]. It 1204
was proposed that the fungal Ecp6 protein containing three LysM domains behaves as a ‘three times one binding site’ when challenged with short chitin polymers in isothermal calorimetry. Nevertheless, cooperativity between LysM domains was suggested when chitooctaose was used as ligand [21]. Recent biochemical and structural data have shown that Ecp6 possesses two binding sites, one of high affinity in the picomolar range, and a second one in the micromolar range [22]. The crystal structure of A. thaliana CERK1 indicates, however, that only one of the three LysM domains is bound to chitin, and that the two other LysM domains play a structural role by maintaining the fold of the extracellular domain. Indeed, in plants and fungi, the multiple LysM domains are linked together by disulfide bonds. In contrast, these bonds are absent in bacteria, and the LysM modules are instead connected by linkers varying in length. Our SAXS data and modelling show that these linkers allow flexibility between the different LysM domains, and may therefore allow each of them to bind a carbohydrate molecule. Whereas our docking and mutagenesis studies show that the CwlS LysM binding site is similar to those of LysM proteins from other phyla, differences in binding strength are observed between LysM proteins from different phyla. Biochemical and/or in vivo binding studies of plant LysM proteins have shown affinities in the low nanomolar range for GlcNAc polymers [8,23–25]. Recently, the affinity for chitin polymers of fungal proteins was investigated. Ecp6 with three LysM repeats has low-picomolar and micromolar affinity for long chitin polymers [21,22]. Single LysM domains of plant and fungal chitinases have also micromolar affinity for chitin polymers [12,15]. These binding studies, FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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together with our binding analysis of bacterial LysM protein, demonstrate that affinities of LysM proteins for their cognate substrate range from low picomolar to high micromolar, and that LysM affinity cannot be predicted from phylogenetics. From our binding studies, we conclude that, in contrast to plant LysM domain-containing receptors, which have to discriminate between PGN, chitin polymers and lipo-oligosaccharide to distinguish between friends and foes [26–28], bacterial LysM domains might not have evolved to acquire high affinity for PGN, or to make distinctions among different GlcNAc polymers: first, because NlpC/P60 proteins are usually secreted in the vicinity of the cell wall, where PGN is highly concentrated, and therefore do not need to have high affinity for substrates; and second, because GlcNAc polymers, like chitin, are not produced in bacteria.
Experimental procedures Generation of plasmids The CwlS gene, excluding the sequence encoding the first 26 residues predicted to be the signal peptide (as determined by the SIGNALP 4.1 Server [29]), was amplified from B. subtilis genomic DNA with Phusion High-Fidelity DNA Polymerase (Finnzymes). The full-length protein, 4LysM and 1LysM_1 were cloned in-frame with the His-tag and S-tag into pET-30 Ek/LIC expression vectors, and the LysM-deleted versions – 3LysM-Cata, 2LysM-Cata, 1LysM-Cata, and Cata – were cloned in frame with the His-tag, S-tag and glutathione-S-transferase-tag into pET-41 Ek/LIC expression vectors (Novagen). 3LysM, 2LysM and 1LysM_4 were cloned in-frame with the His-tag, S-tag and thioredoxin-tag into pET-32 Ek/LIC expression vectors. The CwlS mutant genes, FL-Mut and derivatives were all cloned into the pGS-21a vector (containing both the His-tag and glutathione-S-transferase-tag), purchased from GenScript. A sequence encoding the tobacco etch virus protease cleavage site was introduced into all of the constructs to allow removal of the affinity tags during the protein purification process.
Protein expression and purification All of the protein derivatives were expressed and purified in the same manner. Proteins were expressed in E. coli BL21 CodonPlus RIL competent cells. An overnight culture grown in LB medium supplemented with antibiotics was diluted 100-fold in 12 L of the same medium, and incubated at 37 °C under agitation. When A600 nm reached 0.8, 1 mM isopropyl thio-b-D-galactoside was added to induce protein expression overnight at 20 °C with a rotational FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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speed of 150 rpm. The cells were pelleted by centrifugation at 12 000 g for 20 min, and resuspended in 100 mL of lysis buffer (50 mM Tris/HCl, pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol, 20 mM imidazole, 1 mM benzamidine). The cells were then lysed by sonication, and centrifuged at 30 000 g for 45 min to remove cell debris. All of the different proteins were purified with the same procedures, and all purification steps were performed at 4 °C. First, the crude extract was subjected to affinity chromatography with Ni2+–nitrilotriacetic acid superflow cartridges (Qiagen) pre-equilibrated with lysis buffer. The beads were then washed with high-salt buffer (50 mM Tris/HCl pH 8, 1 M NaCl, 5 mM b-mercaptoethanol, 50 mM imidazole, 1 mM benzamidine), and the proteins were eluted with elution buffer (50 mM Tris/HCl pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol, 500 mM imidazole, 1 mM benzamidine). Then, His-tagged tobacco etch virus protease was added to the eluted protein in a 1 : 50 (w/w) ratio, and the cleavage reaction was allowed to proceed overnight in a 12 000– 14 000-MWCO dialysis tube (6000–8000 MWCO for Cata; Spectrum Labs) against 1 L of dialysis buffer (50 mM Tris/ HCl pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol). For 2LysM protein purification, the NaCl concentration in the lysis, elution and dialysis buffer was 400 mM instead. After dialysis, the solutions were centrifuged at 3320 g for 30 min to remove aggregated proteins. A second round of nickel affinity chromatography was performed, and the cleaved protein was collected in the flow-through. Finally, the proteins were concentrated to 2 mgmL 1 with 10 000MWCO Vivaspin centrifugal concentrators (Sartorius Stedim, Goettingen, Germany), and further purified by size exclusion chromatography with a Superdex75 10/300 GL column (GE Healthcare, Brondy, Denmark) connected to € an Akta purifier system (GE Healthcare). The proteins were eluted with gel filtration buffer (GFB; 50 mM Tris/ HCl, pH 8, 500 mM NaCl, 5 mM b-mercaptoethanol). All recombinant proteins were at least 95% pure after these purification steps (Fig. 2B).
Carbohydrate polymers Soluble fragments of bacterial cell wall were purchased from CeCo Labs (http://www.cecolabs.de). The S. aureus and B. subtilis cell walls were treated with glucosaminidase; therefore, the purified fragments contained only GlcNAc– MurNAc disaccharides linked by the peptide stem (Fig. 3). The PGN fragments had high purity and were free of lipopolysaccharides. Chitin oligomers were purchased from Seikagaku Biobusiness (Tokyo, Japan) and Sigma-Aldrich (Copenhagen, Denmark).
MST Protein interactions with bacterial cell wall fragments (CeCo Labs) and chitin oligomers (Seikagaku Biobusiness and
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Sigma-Aldrich) were measured with MST. Proteins were labelled with the Monolith NT.115 Protein Labelling Kit BLUE (NanoTemper Technologies, Munich, Germany) to achieve a 1 : 1 molar ratio of labelled protein to dye. A titration series was prepared, in which the concentration of the labelled proteins was kept constant at 100 nM, and the concentration of the titrant (octa-N-acetyl-chitooctaose, hexa-N-acetyl-chitohexaose, penta-N-acetyl-chitopentaose, tetra-N-acetyl-chitotetraose, S. aureus tetramer, S. aureus trimer, B. subtilis trimer, or B. subtilis dimer) was varied in thermophoresis buffer (50 mM phosphate, pH 7.5, 1 M NaCl, 0.1% Tween-20). After an incubation period of 20 min, MST measurements were performed at 25 °C on a Monolith NT.115 instrument (NanoTemper Technologies). For each measurement, the laser was switched on for 30 s and off for 5 s. Binding curves were obtained from the thermophoresis plus jump phase, with IR laser power of 20% or 50%. The negative control for each protein was performed with 100 nM labelled protein in thermophoresis buffer in all 16 capillaries under the same conditions mentioned above. Each experiment was repeated at least three times, and an average KD value was calculated after analysis of the data with GRAPHPAD PRISM.
Endopeptidase activity assay An overnight B. subtilis cell culture (ATCC 6056) was grown in 100 mL of LB medium at 30 °C, with a rotational speed of 120 rpm. One millilitre of the overnight culture was grown in fresh LB medium under similar conditions. This procedure was repeated by diluting the overnight culture five times in fresh LB medium, and the cells were used for turbidity assessment when a A600 nm reading of 1.2–1.3 was achieved. All attenuance readings for the assay were obtained with a Jenway 6300 spectrophotometer. The B. subtilis cells in the mid-exponential to late exponential growth phase were diluted with LB medium to obtain a A600 nm reading of ~ 0.9. Each protein (40 lM) in GFB was added to the diluted cells at a 1 : 1 (v/v) ratio, and the mixture was incubated in a cuvette at 30 °C, with a rotational speed of 120 rpm. A A600 nm reading was recorded at regular intervals of 20 min over a period of 140 min. The positive and negative controls were performed by adding 120 lM lysozyme in GFB, or GFB alone, to the diluted cells. The specific activity reflecting three independent experiments corresponds to the loss of A600 nm, and is expressed in lDmin 1lg 1.
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1 M NaCl, 5 mM b-mercaptoethanol, 1% Tween-20). The reaction mixture was incubated for 20 min at 22 °C, and centrifuged at 10 500 g for 45 min. Then, the supernatant was collected, and the insoluble PGN pellet was resuspended in 15 lL of assay buffer. Ten microlitres of supernatant and the resuspended pellet solution were analysed on an 18% SDS/PAGE gel. The results from five experiments were processed with TOTALLAB QUANT software (BioStep GmbH, Jahnsdorf, Germany). The protein densities from the supernatant and pellet were summed, and from these the proportions of protein densities in the supernatant and pellet (averaged over all experiments) were derived and compared. A negative control was performed each time with 10 lg of each protein in the same conditions as mentioned above, without the PGN, to ensure that the proteins were not precipitating on their own. The binding capacity is expressed as percentages of protein present in the pellet and supernatant.
Homology modelling Homology modelling was performed with the automated mode of the Swiss-model server [30]. Several improvements were made to the models, notably by manually adjusting the Ramachandran plot and rotamer residues with COOT [31].
SAXS SAXS data were recorded at 25 °C and at a wavelength of on a prototype of the NanoSTAR camera from Bru1.54 A ker AXS that has been optimized for solution scattering [32]. The acquisition time depended on concentration. Initial data treatment, buffer subtraction and conversion to absolute scale, with water as a standard, were performed with the SUPERSAXS program package (Cristiano Luis Pinto de Oliveira, Institute of Physics, University of Sa˜o Paulo, Sa˜o Paulo, Brazil and J.S.P., unpublished). The intensity is presented as a function of the scattering vector q = 4p sin(h)/k, where k is the wavelength and 2h is the scattering angle. The forward scattering used to determine the molecular mass of the particles in solution was determined with the indirect Fourier transform procedure [33] implemented in the home-written program WIFT [34,35]. Measurements were performed at two different protein concentrations: 4.7 and 1.7 mgmL 1. Rigid body modelling was performed with CORAL [36], with the homology models of the catalytic and LysM domains generated as described above as input.
Insoluble PGN pulldown assay PGN from B. subtilis (Sigma-Aldrich) was dissolved in Mops/NaOH (pH 7) to a final concentration of 10 mgmL 1. Prior to the assay, PGN was heated at 100 °C for 10 min to denature remnant proteases. In a 50lL reaction mixture, 10 lg of each rotein was added to 50 lg of PGN in the assay buffer (50 mM Tris/HCl, pH 8, 1206
Acknowledgements This research was funded by Danish National Research Foundation, grant no. DNRF79. We thank F. Dolman and D. Reid for critical review of the manuscript, and J. Bauer for fruitful discussions. FEBS Journal 281 (2014) 1196–1208 ª 2013 FEBS
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