Insights into pneumococcal fratricide from crystal structure of the modular killing factor LytC

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1 Insights into pneumococcal fratricide from crystal structure of the modular killing factor LytC Inmaculada Pérez-Dorado, Ana González, María Morales, Reyes Sanles, Waldemar Striker, Waldemar Vollmer, Shahriar Mobashery, José L García, Martín Martínez-Ripoll, Pedro García & Juan A Hermoso SUPPLEMENTAL MATERIAL Supplementary Fig. 1: Secondary structure of LytC and comparison of LytC catalytic module with other members of the GH-25 family. (a)

2 (b) (c)

3 (d) Secondary structure of LytC and comparison of LytC catalytic module with other members of the GH-25 family. (a) Topology diagram of LytC (b) Sequence alignment of all choline-binding repeats in LytC. (c) Catalytic modules of LytC (beige), Cpl-1 (green) (PDB code 1h09), Cellosyl (violet) (PDB code 1jfx) and PlyB (cyan) (PDB code 2nw0). (d) Structural superimposition of CM LytC (beige), CM Cpl-1 (green), Cellosyl (violet) and PlyB (cyan).

4 Supplementary Fig. 2: Intermodular interactions in LytC Intermodular interactions in LytC. LytC structure is formed by an N-terminal cholinebinding module, and by a C-terminal catalytic module. The interface between both modules is built by amino acids belonging to the p11 repeat of the CBM and to the CM. The interface is stabilized by hydrophobic and salt-bridge contacts which are represented in Supplementary Fig. 2.

5 Supplementary Fig. 3: Teichoic acid binding at GYMA sites Teichoic acid interactions at GYMA sites. Docked model of the teichoic acid into the GYMA sites as obtained by GOLD 4. Residues building the GYMA site and the teichoic acid moieties bound are labelled. Dashed lines indicate predicted polar interactions. LytC structure presents three GYMA sites that are constituted by six aromatic residues from three consecutive repeats. Three aromatic residues are required for standard choline stabilization (Trp 82, Tyr 117 and Phe 109 in the figure). A Tyr residue (Tyr 105 in the figure) should be involved in polar interactions with the phosphoryl group of PC, the Trp residue (Trp 135) should show extensive stacking interactions with one N- acetylgalactosamine (GalNAc) ring, while the remaining Tyr residue (Tyr 137) should be involved in polar interactions with the other GalNAc sugar Considering the docking experiments, three pentasaccharide teichoic acid units could be attached to one another through one choline residue per unit, connecting the three GYMA sites available in the CBM of LytC.

6 Supplementary Fig. 4: Tyrosine residue acts as the gatekeeper to the PG binding site. Access mechanism to the active site of LytC by the substrate. Tyr407 acts as a gatekeeper in LytC. In the absence of substrate Tyr407 blocks the entrance to the active site (yellow sticks); upon ligand binding, Tyr407 repositions (blue sticks) itself to allow substrate (green sticks) access and binding.

7 Supplementary Fig. 5: Electron density for the PG fragment. Electron density map observed for the pneumococcal PG fragment in the crystallographic complex. Stereoview representation of the PG fragment in complex with LytC. Electron density of the map (2F o -F c ) contoured at 1.0 σ is represented for the PG fragment and is coloured in grey. Atoms of the ligand are in green and protein is in red.

8 Supplementary Fig. 6: Peptidoglycan-binding sites in LytC and Cpl-1 (a) Cpl-1 (b)

9 (c) Peptidoglycan binding-sites in LytC and Cpl-1. (a) Stereoview of the molecular surface of LytC in complex with a pneumococcal PG fragment. The PG-binding sites 1 and 2 (PGBS1 and PGBS2) are highlighted in orange and blue respectively. Positions of the substrate rings are labelled. Lc loop is coloured in dark red. (b) Stereoview of the molecular surface of Cpl-1 in complex with (2S5P)2 (blue sticks) superimposed with the docked model for (2S5P)3 substrate (white sticks) (Supplementary Fig. 5). PGBS1 and PGBS2 are highlighted. (c). Comparison of the amino acid composition of PGBS1 and PGBS2 in Cpl-1 (left) and LytC (right). Main residues involved in substrate binding are drawn as sticks. Network of salt-bridge interactions in Cpl-1 is representd as doted lines.

10 Structural comparison of the catalytic module of LytC with other members of the GH25 family. Structural superimposition of the catalytic module of LytC (CM LytC ) with the structures of the glycosyl hydrolases of the GH25 family reported until now: Cpl-1 1, Cellosyl 2 and PlyB 3, shows that the core of the CM LytC structure is conserved with respect to the other members of the GH25 family (Supplementary Fig. 1), as reflected by the low rmsd values summarized in Supplementary Table 1. Significant differences in the overall structure locate in the following α-helices and loops flanking the central β-barrel: the α1c (residues ), the N-t of the α3c and α3c-β4c loop (residues ), the β4c-α4c loop (residues ), the helices αac and α5c (residues ), the loop β6c-β7c (residues ), the loop β7c-β8c (residues ), and the C-terminal of β8c. Most of these regions are found near the interface between the catalytic module and the cellwall anchoring module in both LytC and Cpl-1 (Supplementary Fig. 3). The β4c-α4c loop (Lc loop) adjacent to the active site, displays significant differences in extension and composition in the case of LytC with respect the other GH25 members. In LytC, this loop is 11-aa longer than in PlyB, 8-aa longer than in Cpl-1 endolysin, and 2-aa longer than in Cellosyl. Sequence alignment of LytC with other members of the GH25 family reveals that Lc is unique among the GH25 family. Supplementary Table 1: Rmsd values obtained from the superimposition of the CM of LytC with other structures of the GH25 family. GH Cpl-1 (CM) Cellosyl PlyB Rmsd (Å) No. Cα atoms

11 Peptidoglycan-binding sites in LytC Further analysis of the CM of LytC shows complete conservation of the active centre of LytC with respect to Cpl-1, Cellosyl and PlyB structures (Supplementary Fig. 4a). The two catalytic residues (D273 and E365) are structurally observed as well as the distance between them, which agrees with the hydrolytic mechanism with inversion of the configuration proposed for this family of hydrolases 1. Two different PG-binding sites have been described in Cpl-1 5. The PG-binding site 1 (PGBS1) is involved in stabilization of the three GlcNAc, MurNAc and GlcNAc rings at positions +1, +2 and +3, and the PG-binding site 2 (PGBS2) within the active site, stabilizing the MurNAc ring at position -1. In this arrangement, a His residue makes a stacking interaction with GlcNAc at -2 and a large groove stabilizes the stem peptide of the MurNAc at position -1 through a network of hydrophobic and salt-bridge interactions. Analysis of the PG-binding channel clearly shows a full conservation of the PGBS1 of LytC with respect to Cpl-1 (supplementary Table2). LytC also presents the mobile tyrosine found in Cpl-1 (Y407 in LytC and Y127 in Cpl-1) which, in both hydrolases, suffers the same rearrangement upon PG binding making accessible the PG-binding channel 5. On the contrary, PGBS2 displays remarkable differences with respect to Cpl-1, Cellosyl and PlyB in: 1) the Lc loop is markedly longer in LytC and does not display sequence homology with respect to other GH of the family, 2) the molecular surface and amino-acid composition in the groove stabilizing the peptide stem at position -1 (Supplementary Fig. 3). LytC lacks the salt-bridge network found in the PGBS2 of Cpl-1, Cellosyl and PlyB, which was proposed to stabilize the peptide stem of the PG (in -1) 5. In LytC, a hydrophobic channel built by three tyrosines and one valine (Y302, Y331, Y369 and V370) is found instead of this salt-bridge network.

12 Supplementary Table2: Structural correspondence of amino-acids of the PG-binding site in Cpl-1, LytC and other GH25 members PGBS1 PGBS2 Glycan stabilization Peptide stabilization Glycan stabilization Peptide stabilization Cpl-1 LytC Cellosyl PlyB E94 E365 E100 E92 Y125 Y405 Y138 Y121 Y127 Y407 T140 H124 K128 R408 A141 _ P129 S409 S142 H125 A151 A428 A163 P143 H164 _ Y153 Y430 W165 Y145 D10 D273 D9 D6 H14 H277 W13 W10 Y59 Y327 Y62 Y58 D92 D363 D98 D90 E94 E365 E100 E92 D182 D467 D198 D171 S12 S275 S11 S8 S13 E276 H12 K9 K34 R299 K33 R29 E37 _ E36 D32 Y41 _ Y40 Y36 F61 Y329 F64 F60 R63 _ R66 R62 D96 _ N102 T84

13 References 1. Monterroso, B., Albert, A., Galán, B., Ahrazem, O., García, P., Martínez-Ripoll, M., García, J.L. & Menéndez, M. Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11, (2003). 2. Rau, A., Hogg, T., Marquardt, R. & Heilgenfeld, R. A new lysozyme fold. Crystal structure of the muramidase from Streptomyces coloelicolor at 1.65 A resolution. J. Biol. Chem. 276, (2001). 3. Porter, C.J., Schuch, R., Pelzek, A.J., Buckle, A.M., McGowan, S., Wilce, M.C., Rossjohn, J., Russell, R., Nelson, D., Fischetti, V.A. & Whisstock, J. C. The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis. J. Mol. Biol. 366, (2007). 4. Jones, G., Willett, P., Glen, R.C., Leach, A.R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, (1997) 5. Pérez-Dorado, I. Campillo, N.E., Monterroso,B., Hesek, D., Lee, M., Páez, J.A., García, P., Martínez-Ripoll, M., García, J.L., Mobashery, S., Menéndez, M. & Hermoso, J.A. Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1. J. Biol. Chem. 282, (2007)

14 Insights into pneumococcal fratricide from crystal structure of the modular killing factor LytC Inmaculada Pérez-Dorado, Ana González, María Morales, Reyes Sanles, Waldemar Striker, Waldemar Vollmer, Shahriar Mobashery, José L García, Martín Martínez-Ripoll, Pedro García & Juan A Hermoso SUPPLEMENTARY METHODS Docking of a TA unit on GYMA site. Docking was carried out using GOLD (Genetic optimization for Ligand Docking) software 1, that uses the Genetic algorithm (GA). This method allows a partial flexibility of protein and full flexibility of ligand. TA unit was extracted from the NMR structure of a TA chain from S. pneumoniae R6 strain 2. One choline residue of the TA unit was fit onto the choline moiety bound at the GYMA site, as observed in the crystal structure. All water molecules and choline moieties were removed from the protein to evaluate the two scoring functions in GOLD software. For each of the 25 independent GA runs, a maximum number of GA operations were performed on a set of five groups with a population size of 100 individuals. Default cutoff values of 2.5 Å (dh-x) for hydrogen bonds and 4.0 Å for van der Waals distance were employed. When the top three solutions attained RMSD values within 1.5 Å, GA docking was terminated. The RMSD values for the docking calculations are based on the RMSD matrix of the ranked solutions. We observed that the best-ranked solutions were always among the first 10 GA runs, and the conformation of molecules based on the best fitness score was further analyzed. 1

15 Computational model of TA chain attached to the CBM of LytC. Models of the LytC structure with the TA chain, based on the NMR structure of a TA from S. pneumoniae R6 strain 2, were constructed as follows: first, a choline residue of the TA was fit onto the choline moiety bound at the binding site; the TA chain was then manually positioned with the program O 3 in order to fit the next choline moiety onto the closest site. To preserve the original conformation of carbohydrate rings in the TA chain, only torsions in the PC residues and in the flexible ribitol moiety were allowed. The model was subsequently energy-minimized using the CNS 4 program. References: 1. Jones, G., Willett, P., Glen, R.C., Leach, A.R. & Taylor, R. Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, (1997). 2. Klein, R.A., Hartman, R., Egge, H., Behr, T. & Fischer, W. The aqueous solution of a lipoteichoic acid from Streptococcus pneumoniae strain R6 containing 2,4- diamino-2,4,6-trideoxy-galactose: evidence for conformational mobility of the galactopyranose ring. Carbohydr. Res. 281, (1996). 3. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. Improved methods for model building in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, (1991). 4. Brunger, A.T. et al. Crystallographic and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, (1998). 2