Structural characterization of the PliG lysozyme inhibitor family

Journal of Structural Biology 180 (2012) 235–242

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Structural characterization of the PliG lysozyme inhibitor family Seppe Leysen a, Lise Vanderkelen b, Kathleen Van Asten a, Steven Vanheuverzwijn a, Veronique Theuwis a, Chris W. Michiels b, Sergei V. Strelkov a,⇑ a b

Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit Leuven, Herestraat 49 bus 822, 3000 Leuven, Belgium Laboratory of Food Microbiology, Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 26 March 2012 Received in revised form 8 May 2012 Accepted 15 May 2012 Available online 24 May 2012 Keywords: Lysozyme Lysozyme inhibitors PliG Crystal structure SAXS

a b s t r a c t Several Gram-negative bacteria protect themselves against the lytic action of host lysozymes by producing specific proteinaceous inhibitors. So far, four different families of lysozyme inhibitors have been identified including Ivy (Inhibitor of vertebrate lysozyme), MliC/PliC (Membrane associated/periplasmic inhibitor of C-type lysozyme), PliI and PliG (periplasmic inhibitors of I- and G-type lysozymes, respectively). Here we provide the first crystallographic description of the PliG family. Crystal structures were obtained for the PliG homologues from Escherichia coli, Salmonella enterica serotype Typhimurium and Aeromonas hydrophila. These structures show that the fold of the PliG family is very distinct from that of all other families of lysozyme inhibitors. Small-angle X-ray scattering studies reveal that PliG is monomeric in solution as opposed to the dimeric PliC and PliI. The PliG family shares a highly conserved SG(x)xY sequence motif with the MliC/PliC and PliI families where it was shown to reside on a loop that blocks the active site of lysozyme leading to inhibition. Surprisingly, we found that in PliG this motif is not well exposed and not involved in the inhibitory action. Instead, we could identify a distinct cluster of surface residues that are conserved across the PliG family and are essential for efficient G-type lysozyme inhibition, as evidenced by mutagenesis studies. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Three major types of lysozymes can be distinguished in the animal kingdom, including chicken (C-) type, goose (G-) type and invertebrate (I-) type lysozymes. They all catalyze the hydrolysis of the b1–4 glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG), the disaccharide building blocks of the peptidoglycan polymers in the bacterial cell wall. This affects the structural integrity of the cell wall leading to osmotic lysis of the bacterium. Hence, lysozyme is an important enzyme in the innate immune system of vertebrate and invertebrate animals (Callewaert and Michiels, 2010). However, bacteria have developed defensive mechanisms against host lysozymes. Some Gram-positive and Gram-negative bacteria are known to chemically modify their peptidoglycan backbone in order to prevent lysis. Typical modifications include O-acetylation or N-glycolylation of NAM and N-deacetylation of NAG and/or NAM (Davis and Weiser, 2011). Another mechanism, thus far only discovered in Gram-negative bacteria, is the production of proteinaceous lysozyme inhibitors. To date, four biochemically distinct families of lysozyme inhibitors have been identified. They are found in the periplasm ⇑ Corresponding author. Address: Katholieke Universiteit Leuven, Herestraat 49 bus 822, 3000 Leuven, Belgium. Fax: +32 16 333469. E-mail address: [email protected] (S.V. Strelkov). 1047-8477/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jsb.2012.05.006

or anchored on the periplasmic side of the outer membrane (Callewaert et al., 2008; Monchois et al., 2001; Van Herreweghe et al., 2010). At first glance, the presence of lysozyme inhibitors in Gram-negative bacteria seems surprising since the outer membrane makes the peptidoglycan layer inaccessible for lysozyme (Abergel et al., 2007). However, the host’s innate immune system produces molecules like lactoferrin that render Gram-negative bacteria more susceptible to lysozyme by permeabilizing the outer membrane (Callewaert et al., 2008). A combination of lysozyme and lactoferrin was indeed shown to be bactericidal for Vibrio cholerae, Salmonella enterica serotype Typhimurium and Escherichia coli, while each protein alone only had a bacteriostatic effect (Ellison and Giehl, 1991). Lately, several studies confirmed that lysozyme inhibitors protect Gram-negative bacteria against lysozyme when their outer membrane is permeabilized (Callewaert et al., 2008; Deckers et al., 2004; Van Herreweghe et al., 2010). The Ivy (Inhibitor of vertebrate lysozyme) family was the first family of lysozyme inhibitors to be discovered (Monchois et al., 2001). This family can be further divided into two subfamilies in which proteins of the first subfamily (Ivy-1) contain a conserved CKPHDC motif while those of the second subfamily (Ivy-2) contain a more variable CExxDxC motif (Abergel et al., 2007). Ivy-1 proteins strongly inhibit C-type lysozyme and they are weakly active against avian G-type lysozyme but not against G-type lysozyme from fish or the urochordate Oikopleura dioica. On the other hand,

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Ivy-2 proteins do not inhibit lysozyme (Clarke et al., 2010; Kyomuhendo et al., 2008; Nilsen et al., 2003). The crystal structures (PDB ID 1GPQ and 1UUZ) of Pseudomonas aeruginosa Ivy-1 and E. coli Ivy-1 in complex with hen egg white lysozyme (HEWL) explain how Ivy-1 proteins inhibit C-type lysozyme: the conserved CKPHDC motif forms a rigid loop which occupies the active site of HEWL. Here, the highly conserved histidine residue makes hydrogen bonds with Glu35 and Asp52 of HEWL (Abergel et al., 2007). Later, a second family of lysozyme inhibitors which specifically inhibit C-type lysozyme was identified. This family was designated as the MliC (membrane-associated lysozyme inhibitor of C-type lysozyme) / PliC (periplasmic lysozyme inhibitor of C-type lysozyme) family (Callewaert et al., 2008). The crystal structure of P. auruginosa MliC (MliC-Pa) in complex with HEWL (PDB ID 3F6Z) showed that MliC/PliC proteins inhibit C-type lysozyme through a double key–lock mechanism in which two conserved regions occupy the active site (Yum et al., 2009). Recently two more families of highly specific lysozyme inhibitors, which were named the PliI (periplasmic lysozyme inhibitor of I-type lysozyme) family and the PliG (periplasmic lysozyme inhibitor of G-type lysozyme) family, were discovered (Van Herreweghe et al., 2010; Vanderkelen et al., 2011). For the PliI family, a crystal structure of Aeromonas hydrophila PliI (PliI-Ah) has been determined (PDB ID 3OD9 (Leysen et al., 2011)) but structural information is still lacking for the PliG family. Here we report the first crystal structures of PliG family inhibitors, including the homologues from E. coli (PliG-Ec), Salmonella Typhimurium (PliG-ST) and A. hydrophila (PliG-Ah). The PliG family has only a low sequence homology to other lysozyme inhibitor families. For example, PliG-Ec has 28%, 12.6% and 5.6% sequence similarity to PliI-Ah, PliC-ST and Ivy-Ec respectively, as determined using the EMBOSS Needle algorithm. In line with that, we found that the fold of the PliG proteins is very different from that of the C and I-type lysozyme inhibitors. The PliG family shares a highly conserved SGx(x)Y sequence motif with the MliC/PliC and the PliI families where it is involved in lysozyme binding. Surprisingly, our mutagenesis and structural data indicate that this motif is not involved in the inhibition of G-type lysozyme. At the same time, we were able to identify another set of conserved PliG residues that are essential for lysozyme binding, as evident from mutagenesis experiments.

2. Materials and methods 2.1. Expression constructs and mutagenesis The constructs based on vector pET28b(+) expressing PliG from E. coli and S. Typhimurium (PT7-pliG-Ec and PT7-pliG-ST) and the pET26b(+)-based expression construct for A. hydrophila PliG (PT7pliG-Ah) were previously described (Vanderkelen et al., 2011). For G-type lysozyme inhibition assays, point mutations were introduced into the construct expressing PliG-Ec using Phusion DNApolymerase (Finnzymes) PCR with two back-to-back annealing primers of which one contained the desired mutation. The primer sequences are provided in the Supplement. Prior to PCR, the primers were phosphorylated using T4 kinase (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s guidelines. Subsequently the PCR product was circularized by ligation using T4 DNA ligase (Fermentas) and transformed into E. coli DH5a cells. To boost protein yields for crystallization trials, the PliG genes were cloned to other expression vectors. The gene coding for PliG-Ec was amplified without its signal peptide using Pfu DNA polymerase (Fermentas) with the primers PliG-Ec-FP and PliG-EcRP. The resulting fragment was transferred to the pETHSUL vector by ligation-independent cloning (Weeks et al., 2007) to generate

construct pETHSUL-pliG-Ec. To produce methionine-containing protein, Leu75 and Leu112 were mutated to Met by two-step PCR-based site-directed mutagenesis using the primers L75M-FP, L75M-RP, L112M-FP and L112M-RP to generate construct pETHSUL-pliG-Ec2M. The gene coding for PliG-ST was amplified without its signal peptide using Verbatim DNA polymerase (Thermo Scientific) with the primers PliG-ST-FP and PliG-ST-RP. The resulting fragment was transferred to the pETHSUL vector by sequence and ligation independent cloning (SLIC) as described by (Li and Elledge, 2007) to generate construct pETHSUL-pliG-ST. The gene coding for PliG-Ah was amplified from pET26b(+) (PT7-pliG-Ah) without the pelB signal peptide with primers PliG-Ah-FP and PliG-Ah-RP. The resulting fragment was transferred to an in-house pETHSUK vector by SLIC cloning generating construct pETHSUKpliG-Ah. 2.2. Expression and purification of wild-type PliG-Ec and PliG-ST The pETHSUL-PliG-Ec construct was transformed into E. coli BL21(DE3). A single colony was used to inoculate 1 ml LB containing 100 lg/ml ampicillin. After 8 h incubation at 37 °C, 500 ll of this pre-culture was used to inoculate 1L ZYP-5052 auto-induction medium (Studier, 2005) containing 100 lg/ml ampicillin and 0.1% V/V antifoam SE-15 (Sigma). The culture was grown overnight at 24 °C until OD600nm = 4.0. At that point, the temperature was decreased to 18 °C and the culture was allowed to grow for another 24 hours. After cell lysis, PliG-Ec was first purified by subtractive nickel-affinity chromatography as described by (Weeks et al., 2007). Here, the column equilibration buffer contained 50 mM sodium phosphate, 250 mM NaCl and 12.5 mM imidazole at pH7.5 while the elution buffer contained 50 mM sodium phosphate, 250 mM NaCl and 250 mM imidazole at pH7.5. Next, PliG-Ec was purified by ion-exchange chromatography on a 5 ml HiTrap SP HP column (GE Healthcare) equilibrated in buffer IEX1 (50 mM HEPES pH7.0). PliG-Ec was eluted with a linear gradient of 0–400 mM NaCl (0-40% buffer IEX2, with buffer IEX2 containing 50 mM HEPES pH7.0 and 1 M NaCl) over 20 column volumes. Fractions containing PliG-Ec were pooled together and dialysed overnight against SEC buffer (10 mM Tris-HCl pH7.5, 0.5 mM EDTA and 250 mM Kcl). Finally, PliG-Ec was purified by size-exclusion chromatography on a Superdex 75 pg 16/60 column (GE Healthcare) equilibrated with SEC buffer. Purified PliG-Ec was dialysed overnight against 10 mM Tris-HCl pH7.5, 0.5 mM EDTA and concentrated to 11 mg/ml using an Amicon Ultra Centrifugation device with a 3-kDa cutoff (Millipore). The protein concentration was determined by measuring absorbance at 280 nm. PliG-ST was purified similarly and concentrated to 20 mg/ml. 2.3. Expression and purification of PliG-Ec2SeM pETHSUL-pliG-Ec2M was transformed into E. coli BL21(DE3). To generate SeMet labeled PliG-Ec2M (PliG-Ec2SeM), 1 ml LB medium containing 100 lg/ml ampicillin was inoculated with a single colony and allowed to grow for 7 h at 37 °C. This culture was used to inoculate 20 ml P-0.5G medium (Studier, 2005) which was incubated overnight at 37 °C. The 20 ml culture was then used to inoculate 1 L PA-5052 auto-induction medium (Studier, 2005) after which the culture was grown for 7 h at 37 °C and 19 h at 30 °C. PliG-Ec2M was then purified as described above for wild-type PliG-Ec. After purification, the protein was concentrated to 13 mg/ml. 2.4. Expression and purification of PliG-Ah PliG-Ah was expressed and purified largely as described for PliG-Ec and PliG-ST. However, for PliG-Ah, the size-exclusion

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column was equilibrated with 10 mM HEPES pH 7.5, 250 mM KCl and 0.5 mM EDTA. The purified protein was then concentrated to 10 mg/ml in the latter buffer and methylated using formaldehyde and dimethylamine–borane complex (DMAB) as described by Chongyun et al. (Shaw et al., 2007). Excess chemicals were removed by buffer exchange against 20 mM Tris–HCl pH 7.5, 250 mM KCl and 0.5 mM EDTA on a superdex 75 pg 16/60 column. Methylation of the N-terminal amine and the side-chains of all six lysine residues was confirmed by mass spectrometry. The methylated PliG-Ah was dialysed overnight against a crystallization buffer containing 10 mM Tris-HCl pH 7.5 and 0.5 mM EDTA and concentrated to 41 mg/ml.

2.5. Crystallization and data collection Commercially available kits were used to screen for crystallization conditions. Hits were further optimized in hanging drops using 24-well XRL plates (Molecular Dimensions) with 500 ll of precipitant solution in the wells. The best crystals of wild-type PliG-Ec were obtained by mixing 1 ll protein solution with 1 ll precipitant solution containing 0.1 M sodium acetate pH5.0, 0.15 M NaCl and 25% PEG6000. PliG-Ec2SeM crystals were grown by mixing 1 ll protein solution with 1 ll precipitant solution containing 0.1 M sodium propionate/sodium cacodylate/BIS-TRIS propane cocktail buffer at pH5.25 (PCB buffer, prepared according to QIAGEN PACT buffer protocol) and 25% w/v PEG1500. PliG-ST was crystallized by mixing 1 ll protein solution with 2 ll precipitant solution containing 0.1 M HEPES pH7.5 and 20% w/v PEG3350. Crystals of methylated PliG-Ah were grown by mixing 1 ll protein solution with 1 ll precipitant solution containing 0.1 M Na cacodylate pH6.5 and 1 M Na citrate. All crystals were cryoprotected in their precipitant solution supplemented with 20% (w/v) glycerol before being flash-cooled in liquid nitrogen. Diffraction data for

PliG-Ec2SeM were collected at the PXIII beamline (Swiss Light Source, Villigen, Switzerland). For wild-type PliG-Ec, PliG-ST and PliG-Ah Ec diffraction data were collected on an in-house Micro-MaxTM-007 HF microfocus X-ray generator (Rigaku). All datasets were indexed and integrated using XDS (Kabsch, 2010) and scaled using SCALA (Evans, 2006). All data collection statistics are summarized in Table 1. 2.6. Structure solution, refinement and validation The structure of PliG-Ec2SeM was solved by SAD phasing using the Auto-Rickshaw server (Panjikar et al., 2005). The model was almost completely built by ARP/wARP (Morris et al., 2004; Perrakis et al., 1999). Phenix.refine (Adams et al., 2010) and Coot (Emsley and Cowtan, 2004) were used in cycles of structure refinement and manual model building respectively. The structure of PliG-Ec2SeM then served as a template to solve the structures of wild-type PliG-Ec, PliG-ST and PliG-Ah by molecular replacement using the programs Phaser (McCoy et al., 2007) or Molrep (Vagin and Teplyakov, 1997) of the CCP4 suite (1994). MolProbity (Davis et al., 2007) was used to analyse the quality of the final models. Secondary structure elements were assigned using the DSSP algorithm (Frishman and Argos, 1995) implemented in Pymol. 2.7. SAXS measurements SAXS data were collected on the X33 beamline (Deutsches Elektronen-Synchrotron, Hamburg, Germany) using 1.5 Å radiation. Scattering data for PliG-Ec (at 1.96 and 6.5 mg/ml) and PliGAh (at 2.98, 5.22, 7.52 and 12.18 mg/ml) in 10 mM HEPES pH 7.5, 0.5 mM EDTA, and 250 mM KCl were recorded from eight exposures of 15 s. PRIMUS (Konarev et al., 2003) was used for averaging of different exposures, buffer subtraction, and merging of low-an-

Table 1 Crystallographic statistics. PliG-Ec2SeM X-ray source Wavelength (Å) Resolution (Å) Space group Cell dimensionsa,b, c (Å) Rsym (%)a, b Rmeas (%)a, c Average I/ra Completeness (%)a No. of unique reflectionsa Redundancya Rwork/Rfree (%) Reflections in the ’free’ set R.m.s. deviations from ideal values Bond lengths (Å) Bond angles (°) No. of chains/asymmetric unit No. of atoms Protein Solvent Average B-factor (Å2) Protein Solvent Ramachandran plot (%) Favoured Outlier Molprobity validation Score Percentile a b c

PliG-Ec

PliG-ST

PliG-Ah

SLS PXIII 0.9793 56.81–1.25 (1.32–1.25) I41 80.32, 80.32, 31.10 4.6 (43.2) 6.6 (49.9) 25.4 (4.3) 99.6 (99.1) 27609 (3941) 10.6 (7.3) 14.1/17.1 2593

1.54 19.69–1.8 (1.90–1.80) P41 83.55, 83.55, 31.16 6.9 (43.7) 7.5 (49.3) 22.2 (3.8) 95.6 (91.1) 19529 (2712) 6.6 (4.6) 16.6/21.3 995

In-house 1.54 19.79–1.78 (1.88–1.78) P212121 45.34, 89.20, 97.64 3.9 (35.1) 5.1 (46.7) 13.8 (1.8) 98.7 (92.3) 38278 (5137) 3.9 (3.6) 18.9/21.2 1915

1.54 19.06–2.02 (2.13–2.02) P3121 76.26, 76.26, 62.44 7.8 (67.8) 8.2 (75.6) 16.6 (2.5) 98.1 (92.3) 13867 (1862) 8.9 (5.1) 18.4/20.7 692

0.01 1.21 1

0.006 0.96 2

0.007 1.05 3

0.008 1.07 1

1981 217

1838 357

2616 362

934 129

18.9 33.2

16.2 28.8

31.9 39,8

35.1 46.7

99.0 0

99.1 0

98.5 0

100 0

1.13 97

1.53 93

1.69 85

1.57 95

Number in parentheses is for the highest resolution shell. Rsym = RhRl|Ihl  |/RhRl, where Ihl is the intensity of the lth observation of reflection h and is the average intensity of reflection h. p Rmeas = Rh| (nh/(nh  1))Rl|Ihl  ||/RhRl, where nh is the number of observations of reflection h.

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gle data from low-concentration samples with high-angle data from high-concentration samples. CRYSOL (Svergun et al., 1995) was used to calculate theoretical scattering curves from crystal structures to fit them to the experimental data. 2.8. Lysozyme inhibition assay Inhibitory activity of wild-type PliG-Ec and its mutants was determined against G-type lysozyme from Atlantic salmon (SalG) in a turbidity assay using Yersinia enterocolitica ATCC9610 cell wall material as described by Vanderkelen et al. (2011). Specific inhibitory activity in inhibitory units (IU) per milligram was measured, where one IU is defined as the amount of inhibitor causing a 50% decrease of lysozyme activity. Inhibitory activity of the mutants was normalized to the WT protein activity. The experiments were repeated three times.

3. Results and discussion 3.1. Crystal structure determination of the PliG homologues Initially, we obtained well-diffracting three-dimensional crystals of the recombinantly expressed PliG protein from E. coli (Table 1). Since no proteins with substantial sequence homology were found in the PDB, the phasing of diffraction data using molecular replacement was not feasible. Instead, we proceeded via singlewavelength anomalous dispersion (SAD) phasing from SeMetlabeled crystals. As PliG-Ec contains no native methionines, we mutated residues Leu75 and Leu112 to methionines. The leucineto-methionine mutations are known to be well-tolerated i.e. typically causing no major changes in the protein structure and stability. Furthermore, a secondary structure prediction using Jpred 3 (Cole et al., 2008) indicated that residues 75 and 112 should

Fig.1. Comparison of crystal structures of the PliG, PliC and PliI family. Panels A, C and E show cartoon representations of PliG-Ec, PliC-ST (PDB entry 3OE3) and PliI-Ah (PDB entry 3OD9), respectively. Panels B, D and F show the corresponding topology diagrams. The location of a common sequence motif SG(x)xY shared by the three families is highlighted. The side chains of serine and tyrosine within the SG(x)xY motifs are shown as sticks.

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reside within b-structural regions. Therefore, they are suitable for introducing methionines that would stay well-ordered in the crystal structure. This prediction was confirmed after the X-ray structure was established (Fig. 1A,B). The obtained SeMet-labeled mutant (PliG-Ec2SeM) yielded crystals in spacegroup I41 which is different to the spacegroup P41 of the WT protein. SAD data to 1.25 Å were collected, followed by a largely automated phasing, model building and refinement with final Rwork = 0.141, Rfree = 0.171. One PliG-Ec2SeM molecule was found in the asymmetric unit. Thereafter the diffraction data for wild-type PliG-Ec were phased by molecular replacement (Table 1). The PliG-Ec2SeM and WT structures are virtually identical, with a Ca-atom RMSD of 0.25 Å. In addition, we have obtained crystals for PliG homologues from S. Typhimurium and A. hydrophila. The crystals of PliG-Ah could only be grown after the solubility of the protein had been decreased via reductive methylation of its N-terminal amine and six lysine residues. Both the PliG-ST and the PliG-Ah structures were phased by molecular replacement using the PliG-Ec2SeM monomer as a search model, at resolutions of 1.8 and 2.0 Å, respectively (Table 1). PliG-ST shares 80% sequence identity with PliG-Ec. In line with this, superimposition of the structures (Fig. 2A) revealed an identical fold with a Ca-atom RMSD of 0.49 Å over 98 paired residues (the residues that deviate by less than 2 Å upon superposition, as calculated by the MatchMaker tool in Chimera (Pettersen et al., 2004)). PliG-Ah shares only 33% sequence identity with PliG-Ec but nevertheless, a structural superimposition (Fig. 2B) revealed that both proteins have the same fold with a Ca-atom RMSD of 0.95 Å over 82 paired residues. The largest structural discrepancy with the protein backbone of PliG-Ec is found in the region constituted by residues 78–101 (corresponding to residues 77–100 on PliG-Ec). The strand b7 is much longer in PliG-Ah (five residues) than in PliG-Ec (two residues). In addition, loops 5 and 6 differ in length and conformation for the two proteins (Fig 2B). The observed conformation of loop 5 in PliG-Ah appears to be affected by a crystal contact with loops 3 and 7 of a symmetry-related molecule (Fig. 2B and Supplementary Fig. 2). Not surprisingly, these structural differences are found in the region of the lowest sequence similarity between the two proteins (Fig. 3). It should be also noted that PliG-Ec and PliG-Ah show similar inhibitory activities (Vanderkelen et al., 2011). Therefore, the region with the structural differences is probably not involved in the inhibition of G-type lysozyme.

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3.2. The fold and oligomerization properties of PliG are distinctly different from those of the MliC/PliC and PliI families Just like the PliC/MliC and PliI family inhibitors, PliG is a predominantly b-structural protein (Fig. 1). Similar to the PliI family, PliG reveals a b-sandwich structure. In PliG-Ec, this sandwich is formed by two sheets containing five strands (b1, b3, b5, b6 and b8) and four strands (b2, b4, b7 and b9) respectively, while the PliI monomer contains 2  4 strands. In contrast, PliC/MliC proteins form an eight-stranded b-barrel. However, the PliG monomer differs radically from the two other families in the fold topology, as evident from the connections between the b-strands (Fig. 1B,D,F). In PliG, the consecutive b-strands alternate between the two sheets (with the exception of strands b5 and b6 that belong to the same sheet). The fold topology may be described as a two-thread, lefthanded solenoid. The first thread of the solenoid begins with b1 and runs through b5 (Fig. 1A). The second thread starts from b6 which is aligned with b5 and follows the same path in the opposite direction all the way through to the last strand b9. Here, b8 inserts in an antiparallel way in-between of b3 and b5, while b9 inserts in an antiparallel way in-between of b2 and b4 (Fig. 1A,B). Just like in the two other families, most adjacent b-strands in PliG are antiparallel. Also, there is also a short a-helix of five amino acids in the loop connecting strands b8 and b9. Compared to PliG, both PliC/ MliC and PliI have a much simpler connectivity of the strands. Each of the two b-sheets of PliI contains four antiparallel strands following each other in a regular way, with an additional C-terminal ahelix of five amino acids (Fig. 1F). The eight strands of the PliC/MliC barrel also follow this perfectly regular antiparallel arrangement, additionally stabilized by a conserved disulfide bond between the first and the last b-strand (Fig. 1D). In order to find out the natural oligomeric state of the PliG proteins we have used small-angle X-ray scattering (SAXS). The scattering curves collected from solutions of the PliG homologues from E. coli and A. hydrophila did not suggest any concentrationdependent oligomerization (in a range of concentrations from 2 to 10 mg/ml). Moreover, the experimental curves could be perfectly fitted by the theoretical scattering calculated from the crystallographic models of the corresponding monomers (Supplementary Fig. 1). In line with this, analysis of the crystal lattice packing for each of the three PliG homologues using the program PISA (Krissinel and Henrick, 2007) did not suggest any stable oligomers. We conclude that PliG exists as a monomer in solution. In

Fig.2. Comparison of PliG structures from different species. (A) Structure alignment of PliG-ST (green) and PliG-Ec (cyan). (B) Structure alignment PliG-Ah (gray) and PliG-Ec (cyan). The view is rotated by 120° about the vertical axis compared to panel A. The region where the two structures deviate the most from each other are highlighted in red (PliG-Ah residues 78–101) and blue (PliG-Ec residues 77–100). The dashed red line marks the position of a crystal contact that may influence the conformation of loop 5 in the PliG-Ah structure. A detailed view of this contact is shown in Supplementary Fig. 2.

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Fig.3. Multiple sequence alignment of PliG homologues. The alignment was created using STRAP (Gille and Frommel, 2001). Residues conserved in >50% of the sequences are highlighted in blue. The secondary structure elements found in the crystal structures of PliG-Ec and PliG-Ah are shown above and below the sequence alignment, respectively. The orange box marks the conserved region containing the SGxY sequence motif. Red asterisks highlight the PliG-Ec residues that were predicted to be part of a protein– protein interface using the meta-PPISP algorithm (Qin and Zhou, 2007). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

contrast, we have shown earlier that both PliI-Ah and PliC-ST form stable dimers in solution (Leysen et al., 2011). Interestingly, however, different oligomerization states were sometimes reported also within the same inhibitor type. For example, a crystal structure (PDB ID 1XS0) and size-exclusion chromatography show that E. coli Ivy-1 forms a homodimer while the crystal structure of its P. aeruginosa homologue (Ivy-1-Pa) in complex with HEWL (PDB ID 1UUZ) indicates that Ivy-1-Pa is a monomeric protein (Abergel et al., 2007; Monchois et al., 2001).

3.3. The conserved SGxY motif of the PliG family is not involved in the inhibition of G-type lysozyme The mechanism of C-type lysozyme inhibition by MliC/PliC proteins was analyzed by Yum et al. (2009), who solved the crystal structure of a complex between MliC-Pa and HEWL revealing a ‘double key–lock’ interaction. Interestingly, two conserved regions, CS1 carrying a SGxxY motif and CS2 carrying a YxxxTKG motif, were identified across the MliC/PliC family. Both these motifs are

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Fig.4. PliG-Ec region involved in the interaction with G-type lysozyme. (A) Crystal structure of PliG-Ec with residues predicted as being part of a protein–protein interface colored in rainbow colors from red to blue, with red indicating the highest probability. The prediction was done using the meta-PPISP algorithm (Qin and Zhou, 2007). The highest scoring residues are labeled. S104 and Y107 of the SGxY sequence motif are also labeled. (B) Inhibitory activity of PliG-Ec mutants towards G-type lysozyme from Atlantic salmon, relative to the wild-type PliG-Ec. For mutants with significantly reduced inhibitory activity, p-values are indicated.

involved in the interactions with lysozyme’s active site, as Ser89 of CS1 makes a hydrogen bond with HEWL Asp 52 and Lys103 of CS2 forms salt bridges with HEWL Glu35 and Asp52. In addition, a shallow pocket on the MliC-Pa surface accommodates a loop of HEWL. This pocket hosts both Tyr93 of CS1 and Thr102 of CS2 which are hydrogen-bonded to HEWL residue Thr47. Sequence analysis showed that both the PliI and PliG families contain a conserved region that is similar to CS1 of the MliC/PliC family, despite a low overall sequence identity between the three families, while no equivalent of the CS2 region is present (Van Herreweghe et al., 2010; Vanderkelen et al., 2011). Specifically, within the CS1 region, both the PliI and the PliG family contain a SGxY motif that is very similar to the functional SGxxY motif of the MliC/PliC family. Recently, we have used site-directed mutagenesis to show that in PliI-Ah the Ser and Tyr residues within this motif are important for the inhibition of I-type lysozyme, most likely through the insertion of loop 6 in the active site (Leysen et al., 2011). By analogy, one could expect that the SGxY motif of the PliG family (Fig. 3) also plays a role in the inhibition of G-type lysozyme. The crystal structure of PliG-Ec presented here reveals that the SGxY motif (including residues S104 and Y107) is located in loop 7 and the beginning of strand b8 (Figs. 1A, 1B and 4A). S104 is exposed on the surface with a considerable solvent accessible area (68 Å2) (Fig. 4A), and could therefore potentially be involved in inhibition. To evaluate this, we have produced the S104A mutant of PliG-Ec. Its inhibitory activity towards the G-type lysozyme from Atlantic salmon was the same as for wild-type PliG-Ec (Fig 4B). Furthermore, our crystal structure shows that the residue Y107 is not exposed on the surface of PliG-Ec. Therefore, it cannot contribute to the interaction with G-type lysozyme (Fig. 4A). The partially buried localization of the the SGxY motif in PliG and the lack of its involvement in lysozyme inhibition are unexpected, given the importance of the SG(x)xY motif in both the MliC/PliC and PliI families. 3.4. Identification of the PliG region involved in the interaction with G-type lysozyme With the three-dimensional structure of PliG in hand, we attempted to predict the region that is involved in the interaction

with G-type lysozyme. We used the meta-PPISP algorithm (Qin and Zhou, 2007) that was recently demonstrated to outperform earlier computational algorithms for the prediction of protein– protein interfaces (Zhou and Qin, 2007). This computation suggested that the interface should involve residues located in loop 2 and the only a-helix of PliG, as well as several adjacent surface-exposed residues in strands b5, b6 and b8 (Fig 4A). As the next step, we evaluated whether each of the predicted interface residues is conserved across PliG homologues (Fig 3). The predicted interface residues Y47, D48, R115, R119 and K120 were found to be nearly absolutely conserved. Correspondingly, we created a series of site-directed PliG-Ec mutants that contained single, double and triple substitutions of Y47, R115 and R119 by alanines. Most of these mutants exhibited a distinct reduction in their lysozyme inhibition capacity compared to the wild-type PliG-Ec (Fig 4B). In particular, the triple mutant Y47A/R115A/ R119A retained only 8% of the inhibitory capacity of the wild type. These results clearly indicate that both Y47 and R119 are important for lysozyme inhibition. In contrast, the R115A mutation on its own did not affect the inhibitory activity. However, this does not fully exclude the possibility that this residue is located on the lysozyme–inhibitor interface. Indeed, a single mutation may be insufficient to significantly disrupt the entire interface, as observed recently for single interface mutants of PliI-Ah (Leysen et al., 2011). Interestingly, PliG from Bordetella avium (PliG-Ba) that contains a threonine and a glutamate in positions corresponding to Y47 and R115 of PliG-Ec respectively (Fig. 3), showed no inhibitory activity against G-type lysozyme in vitro (Vanderkelen et al., 2011). We also note that in this case the R115 equivalent was substituted by a negatively charged residue, and a more drastic effect should be expected than with alanine substitution. In conclusion, our structural and mutational data indicate that the interaction site for the G-type lysozyme critically involves residues in loop 2 and the short a-helix of PliG, all located far away from the conserved SGxY motif (Fig 4A). In the future, a complete understanding of the interaction interface and the inhibitory mechanism could be achieved via the crystal structure determination of a lysozyme–inhibitor complex.

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