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. 2013 Mar;70(6):1113-22.
doi: 10.1007/s00018-012-1184-1. Epub 2012 Oct 21.

Structural basis of bacterial defense against g-type lysozyme-based innate immunity

S Leysen  1 , L Vanderkelen, S D Weeks, C W Michiels, S V Strelkov
Affiliations

Structural basis of bacterial defense against g-type lysozyme-based innate immunity

S Leysen et al. Cell Mol Life Sci. 2013 Mar.

Abstract

Gram-negative bacteria can produce specific proteinaceous inhibitors to defend themselves against the lytic action of host lysozymes. So far, four different lysozyme inhibitor families have been identified. Here, we report the crystal structure of the Escherichia coli periplasmic lysozyme inhibitor of g-type lysozyme (PliG-Ec) in complex with Atlantic salmon g-type lysozyme (SalG) at a resolution of 0.95 Å, which is exceptionally high for a complex of two proteins. The structure reveals for the first time the mechanism of g-type lysozyme inhibition by the PliG family. The latter contains two specific conserved regions that are essential for its inhibitory activity. The inhibitory complex formation is based on a double 'key-lock' mechanism. The first key-lock element is formed by the insertion of two conserved PliG regions into the active site of the lysozyme. The second element is defined by a distinct pocket of PliG accommodating a lysozyme loop. Computational analysis indicates that this pocket represents a suitable site for small molecule binding, which opens an avenue for the development of novel antibacterial agents that suppress the inhibitory activity of PliG.

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Figures

Fig. 1
Fig. 1
Solution properties of SalG (blue curves), PliG-Ec (red) and their complex (green). a Elution profiles (normalized to the absorbance maximum) of the three samples obtained on an analytical SEC column. b Small-angle X-ray scattering data for the three samples normalized by protein concentration. c Corresponding distance distribution functions. d Fits between the experimental SAXS data (black dots) for the three samples and the theoretical scattering curves (coloured lines) calculated from the corresponding crystal structures (PliG-Ec:PDB ID 4DY3, SalG:PDB ID 3MGW and PliG-Ec/SalG complex: this work). The goodness-of-fit (χ 2) values are reported for each curve. The fits for the three samples were displaced vertically for clarity
Fig. 2
Fig. 2
Two conserved regions of PliG-Ec interact with the SalG active site. a Stereo view of SalG (green) and PliG-Ec (cyan) in the complex. PliG-Ec residues Y47 in loop 2 of as well as residues R115 and R119 in helix 1 (red labels) interact with E73, D86 and D97 (purple labels) in the active site of SalG. These residues are located in two highly conserved regions (indicated by red boxes in b). Another conserved region of PliG-Ec (marked with orange here and in b) contains the SGxY sequence motif shown to be important for lysozyme inhibition in the PliC/MliC and PliI families. The SalG loop containing residue D86 is shown here in the ‘inward’ conformation (see Fig. 4 for more detail). b Multiple sequence alignment of PliG homologues. Ec, E. coli (NP_287417.1), ST S. Typhimurium (ACY87940.1), Ss Shigella sonnei (YP_310126.1), Et Edwardsiella tarda (YP_003296035.1), P Photobacterium sp. SKA34 (ZP_01159524.1), Va Vibrio angustum (ZP_01234798.1), D Desulfovibrio sp. FW1012B (ZP_06368489.1), Ba B. avium (YP_785764.1), Ah A. hydrophila (YP_854659.1). Red asterisks mark all PliG-Ec residues that are located on the SalG/PliG-Ec interface. The alignment was created with STRAP [48]
Fig. 3
Fig. 3
PliG-Ec blocks half of the substrate binding site on SalG. a Location of the substrate binding subsites in a distinct cleft of the SalG structure shown in surface representation (green). Here, the NAG molecules (orange) were positioned in the SalG structure by superposing the crystal structures of goose egg-white lysozyme including NAG in subsites B–D (PDB ID 153L) and of Atlantic cod lysozyme including NAG in subsites B–C and E–G (PDB ID 3GXR). b The structure of the PliG/SalG complex, with the molecular surfaces of both components shown. Binding of PliG-Ec (cyan) blocks the subsites B–D on SalG
Fig. 4
Fig. 4
Alternative conformations of SalG residue D86 and PliG-Ec residue R115 in the SalG active site. a A 2Fo-Fc electron density map (gray mesh) indicates two possible orientations for both residues, colored in different shades of green and cyan, respectively. An anomalous difference map calculated from the long-wavelength data (yellow mesh) reveals the location of the chloride ion (yellow sphere). b The ‘inward’ conformation of the loop containing D86 (45 % probability) occurs in the absence of the chloride ion, with the D86 residue interacting with Yr47, R115, and R119 of PliG-Ec. The black dashed lines mark hydrogen bonding and ionic interactions. c The ‘outward’ conformation of the loop and the D86 side chain (55 % probability) is stabilized by the chloride ion binding
Fig. 5
Fig. 5
Candidate binding site for PliG suppressor compounds. a In the crystallographic complex, SalG (green) is shown in cartoon representation while PliG-Ec (cyan) is shown in surface representation. Residues R99 and Y100 (shown as yellow sticks) of SalG loop 6 insert into a pocket (gray) on the PliG-Ec surface. The catalytic side chains E73, D86, and D97 of the lysozyme are shown in magenta, while the PliG-Ec residues R115 and R119 are in red (compare with Fig. 2a). b Detailed view of the interactions in the pocket, shown in stereo. Both proteins are shown in cartoon representation with the same colors as in (a). The main-chain carbonyl oxygen of SalG R99 is H-bonded to the side chain of PliG-Ec R110, while the side chain of SalG R99 is H-bonded to the main chain carbonyl atoms of both PliG-Ec Y47 and Q113. Y100 makes hydrophobic interactions with PliG-Ec Y74 and L112 through its ring atoms, while its hydroxyl group makes an H-bond with PliG-Ec D72, and its main chain carbonyl oxygen makes an H-bond with the hydroxyl group of the S83 side chain
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