Editor's evaluation: Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins (2021)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development. Editor's evaluation In this report, the authors employed a wide array of biophysical tools and techniques to study the interaction of LukE toxin with chemokine receptors. Although follow-up studies will be required to substantiate all of the conclusions drawn by the authors, the paper will be of general interest as it provides insights into the molecular recognition mechanism of an important toxin interacting with cellular receptors. https://doi.org/10.7554/eLife.72555.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Staphylococcus aureus (SA) is a major opportunistic human pathogen that causes a wide range of clinical manifestations (Tong et al., 2015), and poses growing health concern due to the emergence of multidrug resistant strains. Pathogenic SA strains produce a number of wall-associated or secreted virulence factors that promote growth, nutrient acquisition and evasion of the host immune system (DeLeo et al., 2009; Oliveira et al., 2018). Among this arsenal of virulence factors, the bicomponent leukotoxins are β-barrel pore forming toxins that play an important role in SA pathogenesis by targeting host leukocytes and erythrocytes (Spaan et al., 2017), and appear to be promising targets for drug development (Kong et al., 2016). Human clinical SA isolates can produce up to five different leukotoxins: γ-hemolysin AB and CB (HlgAB and HlgCB), Leukocidin ED (LukED), Panton-Valentine Leukocidin (PVL or LukSF-PV), and Leukocidin AB (LukAB, also known as LukGH). Each leukotoxin is made up of two subunits of approximately 30 kDa: a host cell targeting S-component (HlgA, HlgC, LukE, lukS-PV, and LukA/G), and a polymerization F- component (HlgB, LukD, lukF-PV, and LukB/H), which can bind the plasma membrane through highly conserved phosphocholine binding sites (Olson et al., 1999; Liu et al., 2020). S- and F-components typically display relatively high conservation within each group ( ~ 70% sequence identity), but sequence identity drops below 30% between groups (Spaan et al., 2017). With the exception of LukGH, these toxins are secreted as monomers that bind target cells and subsequently octamerize into a lytic pore on the plasma membrane, composed of alternating S- and F- subunits (Olson et al., 1999; Guillet et al., 2004; Yamashita et al., 2011; Yamashita et al., 2014; Nocadello et al., 2016; Trstenjak et al., 2020). Each toxin is composed of two domains: a central β sandwich called the CAP domain and involved in inter-protomer interactions, and a RIM domain which contains several divergent loops driving host cell specificity. The STEM region is a subdomain of the CAP, which changes its conformation upon pore formation to associate with neighboring protomers and form a β barrel that inserts into the cell membrane (Yamashita et al., 2011; Yamashita et al., 2014). Over the last decade, membrane protein receptors have been identified for most of the bicomponent leukotoxins, providing key insights into their role in SA pathogenesis and the mechanisms driving their cellular tropism and species specificity (Spaan et al., 2017). Specifically, HlgA and LukE were shown to share atypical chemokine receptor 1 (ACKR1) as their main receptor on erythrocytes (Spaan et al., 2015), while recruiting CCR5 (LukE), CCR2 (HlgA), CXCR1, and CXCR2 (both HlgA and LukE) to differentially target specific leukocyte populations (Alonzo et al., 2013; Reyes-Robles et al., 2013; Spaan et al., 2014). Conversely, HlgC and LukS-PV were found to bind monocytes and neutrophils through C5aR1 and C5aR2 (Spaan et al., 2013; Spaan et al., 2014). All of the identified receptors belong to the chemokine or complement receptor families of class A G-protein-coupled receptors (GPCRs), except for the divergent lukGH toxin which binds CD11b (aka complement receptor 3) expressed on phagocytic cells (DuMont et al., 2013). Recently, F-component-specific receptors have been identified as well: human CD45 was shown to act as a receptor for lukF-PV (Tromp et al., 2018), while HlgB was found to interact with ACKR1. Several of these toxin-GPCR pairs have been characterized at the molecular level by swapping mutagenesis of divergent regions/loops (DRs) of the RIM domain, in particular for LukE and HlgA that can target multiple chemokine receptors (Reyes-Robles et al., 2013; Laventie et al., 2014; Tam et al., 2016; Peng et al., 2018; Vasquez et al., 2020). These studies highlighted the critical role of several DRs located in the rim domain in determining receptor specificity. From the receptor’s side, residues of CCR5 were identified in eCL2 and in the upper part of TM7 that lead to severe loss of LukED activity (Tam et al., 2016). Several residues of ACKR1 were also identified by mutagenesis, the most critical being the sulphated tyrosine sTyr41 for which the alanine substitution nearly abolished LukED and HlgAB activity on erythrocytes (Spaan et al., 2015). Interestingly, all of the GPCRs that bind leukotoxins possess between 1 and 4 potentially sulfated tyrosine residues in their N-terminal regions, and the interaction between LukS-PV and the C5aR1 N-terminal region was shown to require tyrosine sulfation (Spaan et al., 2013). In addition, post-translational modification pathways involved in the sulfation of the leukotoxin receptors were recently found to impact on HlgAB, HlgCB, LukED, and PVL induced cytotoxicity (Tromp et al., 2020). Despite these advances, a structural understanding of toxin–chemokine receptors interactions is still lacking due to the unavailability of high resolution structures of the complexes. In the present study, we provide a detailed characterization of the structure and dynamics of monomeric LukE using integrated structural biology methods. We show that recombinant LukE is able to displace bound chemokines from ACKR1, CCR5, and CCR2 on the cell surface using a time-resolved fluorescence energy transfer (TR-FRET) based approach. We next solved the crystal structures of LukE in complex with a small molecule mimicking a sulfotyrosine side chain (p-cresyl sulfate) and with sulfopeptides derived from the N-terminal region of ACKR1 and CCR2, revealing two conserved sulfotyrosine binding sites. Finally, we use computational docking and molecular dynamics (MD) simulations to propose models of the ACKR1-LukE and CCR5-LukE complexes that are supported by previous pharmacological experiments as well as novel mutagenesis data described here. Results Solution and crystallographic structures of LukE The crystal structure of monomeric LukE has previously been reported at 3.2 Å resolution (PDB code 3ROH) (Nocadello et al., 2016). While attempting to co-crystallize LukE in complex with peptides encompassing the sulfated ACKR1 Tyr41 residue, we serendipitously obtained two additional apo crystal forms of LukE at 1.5 Å (apo1) and 1.9 Å (apo2), allowing us to build a higher quality model (see Table 1 and Materials and methods). The apo2 structure, like the original 3ROH structure, belongs to space group I4, while the apo1 structure crystallized in P212121 and features an entirely different crystal packing (Figure 1A and Figure 1—figure supplement 1). All three structures contain one copy of the protein in the asymmetric unit and display very similar conformations, with Cα RMSDs of 1.18 Å (apo1) and 0.81 Å (apo2) relative to the 3ROH structure (Figure 1A). One notable difference is the presence of residues 12–23 belonging to the N-terminal signal sequence in apo2, which are involved in crystal packing interactions, possibly accounting for the higher resolution compared to the 3ROH structure (Figure 1A and Figure 1—figure supplement 1). A comparison of the B-factors of the three structures is shown in Figure 1—figure supplement 1D-F, highlighting the influence of crystal packing on protein flexibility within the crystals. Table 1 X-ray data collection and refinement statistics (molecular replacement). Apo1Apo2p-cresol sulfate soakAcDsYDsYG-NH2 soakAcDSFPDGDsY GANLE-NH2 soakData collection Space groupP212121I4P212121P212121P212121Cell dimensions a, b, c (Å)62.58, 72.20, 78.81135.79, 135.79, 63.5362.58, 73.49, 79.3463.30, 72.41, 79.0062.97, 71.18, 79.23a, b, g (°)90.00, 90.00, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.0090.00, 90.00, 90.00Resolution (Å)49.01–1.46 (1.48–1.46)48.01–1.90 (1.94–1.90)47.65–1.60 (1.63–1.60)49.40–1.40 (1.42–1.40)47.16–1.55 (1.58–1.55)Rmerge0.079 (2.601)NA0.100 (1.510) NA0.040 (0.710)0.038 (1.129) NA0.032 (0.811)CC1/21.000 (0.508)0.984 (0.748)0.996 (0.624)0.999 (0.483)0.998 (0.622)I / σI17.2 (1.0)19.2 (1.9)15.5 (1.7)15.8 (1.2)17.5 (1.5)Completeness (%)99.9 (98.5)100 (100)99.3 (99.2)99.2 (97.3)99.2 (99.8)Redundancy13.1 (12.0)13.7 (13.8)3.4 (3.1)4.1 (3.6)3.2 (3.3)Refinement Resolution (Å)49.1–1.4648.1–1.9047.7–1.6040.9–1.4047.2–1.55No. reflections62,77545,73448,96772,13652,377Rwork / Rfree17.81/19.4118.63/20.3018.12/20.4618.44/19.8118.12/20.37No. atoms Protein2,3702,4722,3512,3592,276Ligand/peptide005771140Water328323260293215B-factors Protein28.337.532.831.736.9Ligand/peptideNANA29.631.838.3Water44.353.348.447.851.1R.m.s. deviations Bond lengths (Å)0.0090.0090.0100.0100.010Bond angles (°)1.1081.0921.1021.2261.163< Ligand occupancy>NANA0.670.590.51 Values in parentheses are for highest-resolution shell. NA-not applicable, Rmerge value over 1 is statistically meaningless. Figure 1 with 2 supplements see all Download asset Open asset Solution and crystallographic structures of leukotoxin LukE. (A) The crystal structures of LukE in P212121 (Apo 1, green) and I4 (Apo 2, cyan) space groups are shown in cartoon representation and overlaid onto the previously published crystal structure of LukE (magenta), also in I4 space group (PDB code 3ROH). (B) Optimized ensemble of 10 models corresponding to the fitted SAXS profile in C. The N-terminal extension belonging to the signal peptide and the C-terminal polyhistidine tag are shown in orange and black, respectively. The CAP, RIM, and STEM domains are colored in cyan, deep blue and light blue, respectively. Other regions of interest are also indicated such as the D-region, and divergent loops 1–4. (C) Small angle x-ray scattering (SAXS) profiles of LukE measured at 1, 2, and 4 mg/ml are shown as black, red and green lines, respectively. The merged SAXS curve is shown as a brown line with the fitting curve obtained using the ensemble optimization method (EOM) in orange. (D) Radius of gyration distributions for the initial pool ensemble (gray area and black line) and for the optimized ensembles (red bars) obtained using the merged SAXS curve. In order to obtain structural information about LukE directly in solution, we used SAXS. The experimental SAXS profiles are shown in Figure 1B. The samples were free from aggregation as evidenced by the linearity of the Guinier region, however, the measured radius of gyration (Rg) increased from 2.41 ± 0.02 nm at 1 mg/ml to 2.68 ± 0.08 at 4 mg/ml of protein, suggesting a slight tendency for interparticle attraction. This was consistent with a native mass spectrum of LukE showing a small proportion of dimeric forms in equilibrium with LukE monomers (Figure 1—figure supplement 2). In order to extract structural information from the SAXS data, we turned to the ensemble optimization method (EOM) using ensembles of models derived from atomistic MD simulations (Figure 1C, D, and Figure 1—figure supplement 1G-I). The MD generated models of LukE were fitted to a merged SAXS curve to remove the interparticle attraction effects (Figure 1B), yielding a χ value of 0.79. Rg values of the selected models were in a narrow range between 2.40 and 2.55 nm (Figure 1D), consistent with the values of 2.32 and 2.43 nm calculated from the apo1 and apo2 structures. Conformations of the models from the optimized ensemble show a very rigid core, with highly flexible terminal regions corresponding to the signal peptide and polyhistidine tag. Localized flexibility is also present in the divergent loops of the RIM domain, and in the STEM region loop (residues 151–159) (Figure 1B and Figure 1—figure supplement 1). The three β strands of the STEM remain, however, relatively rigid, suggesting that specific conformational triggers are required to explore the extended conformations observed in leukotoxin pore structures. LukE competes with CCL5 binding onto ACKR1 and CCR5, but also with CCL2 binding onto CCR2 We next sought to analyze the binding of LukE to its human chemokine receptor targets. A wealth of experimental information is available indicating that the expression of CCR5, ACKR1, CXCR1, and CXCR2 render human cells susceptible to LukED killing (Alonzo et al., 2013; Reyes-Robles et al., 2013; Spaan et al., 2015; Tam et al., 2016; Vasquez et al., 2020). Direct interaction between LukE and CCR5 or ACKR1 has also previously been characterized by surface plasmon resonance (SPR) in purified systems, indicating Kd values around 40 and 60 nM, respectively (Alonzo et al., 2013; Vasquez et al., 2020). In order to complement the available experimental data and to verify that our recombinantly produced LukE is biologically active, we performed competitive binding assays in HEK293T cells using Homogenous TR-FRET technology (Zwier et al., 2010; Figure 2). For these experiments, we selected ACKR1 and CCR5, which are major leukotoxin receptors in erythrocytes and leukocytes, but also CCR2 which is normally an in vivo target of HlgA rather than LukE (Spaan et al., 2014). SNAP-tag-fused CCR5, ACKR1, and CCR2 receptors transiently expressed in HEK293 cells were covalently labeled with Lumi4-terbium as donor. Cells were then incubated in the presence of 5 nM tracer chemokines d2-CCL5 (CCR5 and ACKR1) or d2-CCL2 (CCR2). Addition of increasing concentrations of LukE led to a decrease in the TR-FRET ratio for all receptors indicating the ability of LukE to bind competitively with IC50s of 231.7 nM ±0.19, 230.4 nM ±0.14 and 64.2 nM ±0.14 at CCR5, ACKR1, and CCR2, respectively. These results indicate at least partial overlap between the LukE and CCL2/5 binding sites on the receptors extracellular surfaces and are consistent with previous observations regarding ACKR1- and CCR5- LukE interactions (Alonzo et al., 2013; Spaan et al., 2015). Interestingly, LukE inhibited CCR2–CCL2 interaction with a better IC50 than observed for CCR5–CCL5 and ACKR1–CCL5 pairs, suggesting a strong interaction between CCR2 and LukE. Figure 2 Download asset Open asset Binding of LukE in live cells as determined by competition time-resolved fluorescence energy transfer (TR-FRET). The upper panel shows a schematic of the competitive TR-FRET assay. Addition of toxins disrupts energy transfer between a SNAP-tagged receptor labeled with a Terbium donor and a d2-chemokine acceptor (Zwier et al., 2010). The bottom panel shows the competition dose-response curves at receptors CCR5, ACKR1, and CCR2. 5 nM tracer ligands, CCL5-d2 for CCR5 and ACKR1 and CCL2-d2 for CCR2 were used to determine TR-FRET at their respective receptors in the presence of LukE. IC50 values are quoted in-text. Data shown is mean ± SEM of three independent experiments performed in triplicate. Figure 2—source data 1 Raw data of the LukE competition TR-FRET assay. https://cdn.elifesciences.org/articles/72555/elife-72555-fig2-data1-v4.xlsx Download elife-72555-fig2-data1-v4.xlsx Crystal structure of LukE in complex with p-cresyl sulfate reveals three potential sulfotyrosine binding sites In order to obtain structural insights into the interaction of LukE with its receptors, we carried out crystal soaking experiments using apo1 LukE crystals. Because recognition of sulfated tyrosines appears to be a common trend among leukotoxins, we selected 3 synthetic molecules for soaking, in an attempt to identify potential sulfotyrosine binding sites: (1) para-cresyl sulfate (pCS) is a metabolite of tyrosine and a uremic toxin (Gryp et al., 2017). Its chemical structure is identical to that of a sulfotyrosine side chain (Cβ replaced by a methyl group); (2) the 34DSFPDGDsYGANLE46 peptide of ACKR1, with a functionally critical sulfated tyrosine in position 41; and (3) the 25DsYDsYG29 peptide of CCR2 bearing 2 sulfated tyrosines in position 26 and 28. Although CCR2 is normally an in vivo target of HlgA rather than LukE, Tam and coworkers have shown that cells expressing WT CCR5 or a CCR5 chimera harbouring the CCR2 N-terminal region display the same susceptibility to LukED intoxication (Tam et al., 2016). This interchangeability of the N-terminal region is perhaps not so surprising given that both sequences contain multiple tyrosine residues that can be efficiently sulfated (Farzan et al., 1999; Preobrazhensky et al., 2000; Tan et al., 2013). Crystals soaked with all three data that revealed strong additional corresponding to the (Figure supplement 1 and Figure supplement 2). The structure of LukE is shown in Figure molecules interact with LukE at which we 1, 2, and 1 forms a surface pocket with at the bottom and in a with the sulfate group (Figure and groups the sulfate and the the of and The upper part of the pocket is by and which the interacting from the These residues to and in HlgA and are part of the that an important role in activity and Peng et al., 2018). Figure 3 with 2 supplements see all Download asset Open asset Crystal structure of LukE in complex with p-cresyl (A) of the asymmetric unit of the is shown in cartoon representation and colored The sulfate molecules are shown in with protein in of the sulfotyrosine binding sites. The code is the same as in (A) with sulfate molecules and protein shown in between and protein are shown as In protein residues from an interacting molecule are shown in cartoon and 2 is a relatively area located approximately nm below 1 (Figure The sulfate is by with and and its group the in and from 1 are also involved in with the from a molecule binding through with the sulfate 3 to a small in between and (Figure The molecule is by interactions of the sulfate group with with and as well as between the and the part of Crystal structures of LukE in complex with the sulfated peptides 34DSFPDGDsYGANLE46 of ACKR1 and 25DsYDsYG29 of CCR2 show sulfotyrosine binding at sites 1 and 2 The structure of LukE in complex with the ACKR1 peptide 34DSFPDGDsYGANLE46 is shown in Figure we found two bound of the which be due to the potentially very high peptide used crystal soaking experiments (see Materials and methods). and residues are for the and respectively. Although the signal for each sulfotyrosine is the of the peptide residues is for the peptide (see in Figure This is in the which are for the but for the peptide The first peptide with 1 through its sTyr41 in a similar to the The residues the of the binding the surface area of and provide additional through multiple interactions (Figure and and of LukE. forms a with LukE and is to from The peptide sTyr41 with 2, in a that is also similar to a different conformation and an additional is between the sulfate and (Figure Other interactions several between and and interactions between and and of LukE. Although the conformation of both peptides is by multiple interactions with LukE, it be that the peptide chain with its from the location of the In addition, the is relatively for the and the conformation of the peptides is by crystal packing and the Figure 4 Download asset Open asset Crystal structures of LukE in complex with ACKR1 peptide 34DSFPDGDsYGANLE46 and CCR2 peptide (A) of the asymmetric unit of the ACKR1 is shown in cartoon representation and colored in The two of the peptide with their respective sulfotyrosine bound to 1 and 2 are shown in and green cartoon and protein are shown as (B) of the asymmetric unit of the CCR2 is shown in cartoon representation and colored in The CCR2 peptide is shown in cartoon and protein are shown as and of the sulfotyrosine binding 1 in the ACKR1 (C) and CCR2 (D) crystals. The code is the same as in (A) and (B) with peptide and protein shown in between and protein are shown as and of the sulfotyrosine binding 2 in the ACKR1 and CCR2 crystals. residues from an interacting molecule are shown in cartoon and The structure of LukE bound to the CCR2 peptide 25DsYDsYG29 is in Figure We found that and of CCR2 respectively bind to 1 and 2 through the same interactions observed in the and ACKR1 structures (Figure conformations were for by a of approximately of the (Figure and Figure supplement 1). was still present model and the of a conformation of with its sulfate group interacting at 2 (Figure supplement 1C, This conformation with the sulfate group of suggesting the of a of molecules in which while its to the This is consistent with the for 2 1 sulfotyrosine and and the presence of a chain at in the (Figure supplement 1). These observations indicate that the binding of the sulfated CCR2 peptide is highly within the simulations of LukE onto ACKR1 and CCR5 In order to these crystallographic results into a we out to build computational models of the ACKR1-LukE and CCR5-LukE complexes. A wealth of mutagenesis data is available in the that provides partial information about interacting residues for the ACKR1-LukE (Spaan et al., 2015; Peng et al., 2018; Vasquez et al., and CCR5-LukE complexes (Reyes-Robles et al., 2013; Tam et al., 2016). The regions of LukE that are potentially in receptor interaction based on previous work and on the structures were onto the structure of LukE (Figure revealing a interaction Figure 5 Download asset Open asset docking of ACKR1-LukE and (A) of the LukE residues potentially involved in receptor LukE is shown in surface and cartoon representation and colored in 1 and 2 sulfotyrosine binding sites are colored in orange. Other residues that are potentially involved in receptor binding are colored in based on previous mutagenesis work and Reyes-Robles et al., 2013; Tam et al., 2016; Peng et al., 2018; Vasquez et al., 2020). (B) and mean deviations relative to the for the ACKR1-LukE (B) and CCR5-LukE (C) docking The identified by is shown in (D) and ACKR1-LukE (D) and CCR5-LukE models selected from of the N-terminal region (residues of ACKR1 and residues of energy and MD The receptor is shown in cartoon representation and colored in green (ACKR1) or light tyrosines are shown as The of docking LukE onto ACKR1 and CCR5 was performed in two in the first the toxin was onto the experimental structures or a model ACKR1) the N-terminal region of the using the docking mutagenesis data were into by providing relatively strong on the of the bound toxin (see Materials and methods). In the the N-terminal region of the receptor was to the model of each and multiple MD simulations were performed in order to the N-terminal region of the receptor onto the toxin and the of the docking Figure show the to the for the ACKR1-LukE and CCR5-LukE docking revealing in both a deep energy corresponding to the The ACKR1-LukE and CCR5-LukE are also the with and respectively. The for each complex is shown in Figure of the N-terminal residues and to MD The relative of LukE in each complex is with LukE forming a angle with the membrane compared to the ACKR1-LukE (Figure in both LukE loop 4 inserts into the pocket and with ACKR1 and while loop 3 with the position of and the N-terminal region of the receptor for both pairs with sulfotyrosine interactions at 1 2 observed in crystal structures. ACKR1 is located 10 residues of that the of while the potentially sulfated tyrosines of CCR5 and are located residues of the at the of CCR5 This between and the sulfated with the known location of 1 and 2 on LukE, provide an additional