Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota1,2. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate1,3, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.
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Electron Microscopy Data Bank
Protein Data Bank
Coordinates of the crystallographic structure of active human RegIIIα have been deposited in the Protein Data Bank with accession code 4MTH. The cryoEM map has also been deposited in the 3D EM database under accession code EMD–5795.
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We thank T. Craig for assistance with the liposome disruption assays. The MODE-K cell line was provided by D. Kaiserlian, INSERM U851, Lyon, France. We thank E. Egelman for sharing his programs and for offering advice on cryoEM data analysis. This work was supported by NIH R01 DK070855 (L.V.H.), NIH R01 GM088745 and GM093271 (Q.-X.J.), NIH R01 NS40944 (J.R.), Welch Foundation (I-1684 to Q.-X.J.), NSF CAREER MCB0845286 (M.G.), a Helen Hay Whitney Fellowship (S.M.), a Burroughs Wellcome Foundation New Investigators in the Pathogenesis of Infectious Diseases Award (L.V.H.), and the Howard Hughes Medical Institute (L.V.H.). Part of this work was performed in laboratories constructed with support from NIH grant C06 RR30414.
The authors declare no competing financial interests.
Extended data figures and tables
An overall model that incorporates both the peptidoglycan and lipid-binding functions of RegIIIα is depicted. Combining our current and previous findings, we propose that RegIIIα recognizes and kills its bacterial targets in two distinct steps. First, RegIIIα is secreted from epithelial cells as a soluble monomer that recognizes Gram-positive bacteria by binding to peptidoglycan carbohydrate via an EPN motif located in the long loop region1,3. Second, RegIIIα kills bacteria by oligomerizing in the bacterial membrane to form a hexameric membrane-penetrating pore that is predicted to induce uncontrolled ion efflux with subsequent osmotic lysis. The inhibitory N terminus of pro-RegIIIα hinders lipid binding and consequently suppresses pore formation until it is removed by trypsin after secretion into the intestinal lumen4. We propose that the inhibitory N-terminal peptide evolved to minimize collateral damage from the RegIIIα pore-forming activity during RegIIIα storage in the membrane-bound secretory granules of epithelial cells. In support of this idea, RegIIIα damages mammalian cell membranes and the N-terminal pro-segment limits this toxicity (Fig. 4d, e).
a, b, Impact of NaCl concentration on RegIIIα membrane permeabilization activity. a, 10 μM RegIIIα was added to liposomes (100 μM lipid) in the presence of varying NaCl concentrations. Representative results are shown. b, Averaged results from three independent replicates of the experiment shown in a. c, Pro-RegIIIα does not inhibit RegIIIα bactericidal activity. 10 μM of purified recombinant pro-RegIIIα, RegIIIα, or a combination of the two was added to ∼105 c.f.u. of L. monocytogenes for 2 h at 37 °C. Surviving bacteria were quantified by dilution plating.
Analysis of RegIIIα conductance in lipid bilayers. The trace of a typical single channel recording gave rise to the event histogram shown here. At −80 mV, there was a short latency before the first opening event, which led to the baseline current of −6.5 pA at −80 mV. The baseline current was subtracted so that the baseline corresponds to a peak at 0 pA. Once we assigned two basic peaks at −53 pA and −81 pA as two independent opening events (i1 and i2), all the other major peaks in the histogram are linear combinations of these two basic events (as labelled). The data therefore suggested two different scenarios. One is that there are three pores, and each pore has two different conducting states, which may reflect the flexible diameter of the pore. The other is that i1 and i2 reflect two different pores that have different diameters, and that there are at least five different channels in the membrane to produce the observed histogram. This second scenario correlates with the observed variability in helical symmetry. With the idea of variability and protein dynamics in mind, it is likely that the two types of pores may interconvert with each other in the membrane. From the basic events, we estimated the pore diameters by applying the Nernst–Planck equation. In the experimental conditions, our recording chambers had 150 mM K+, 25 mM Na+, 215 mM Cl−, 20 mM Mg2+ and 10 mM MES pH 5.5 in the cis side, and 20 mM K+, 25 mM Na+, 45 mM Cl− and 10 mM MES pH 5.5 in the trans side. The reversal potential (EK, ENa, ECl and EMES) for each ion could be calculated (EK = 50.9 mV, ENa = 0 mV = EMES, and ECl = −39.5 mV). In the trans side, there is a trace amount of Mg2+ (∼10 μM), which gives a reversal potential EMg of 92 mV. Our dye leakage assay showed that the pore was open at Vmem = 0 mV transmembrane potential, ruling out significant voltage-dependent gating of the RegIIIα channel. On the basis of the ion replacement studies we did for different ions, we estimated the relative permeability of different ions to be: PK = PNa = 1.0; PCl = 0.85; PMES = 0.73 and PMg = 0.66. The measured relative permeation rates showed that the pore has very weak cation selectivity, and favours K+/Na+ over Mg2+ due to the charge density difference. Under the same assumption, the average conductance (<g>) of the two basic opening events (i1 and i2) could be calculated as the following:
The two calculated conductance levels of 100 pS and 152 pS were then entered into the Nernst–Planck equation for electrodiffusion and gave rise to an approximate estimate of the pore diameter of 12 Å and 14 Å, respectively, which is in good agreement with the observed pore size in the reconstructed three-dimensional structure of the pore (Fig. 3b). A more rigorous calculation of the ion flux is possible with a high-resolution picture of the potential profile, but is beyond the scope of this paper.
a, Negative staining EM controls lacking RegIIIα or liposomes are shown. b–d, RegIIIα pore complexes assemble into filaments. b, RegIIIα forms filaments in the presence of lipid vesicles. 20 μM RegIIIα was incubated for 2 or 20 min with vesicles composed of PC/PS (85%:15%). Samples were visualized by transmission electron microscopy. Grids were stained with anti-RegIII antibody1,10 to confirm that the filaments were composed of RegIIIα. Filamentation required membranes, as no filaments were observed in the absence of liposomes. Arrows indicate examples of filaments in each image. c, 20 μM RegIIIα carrying a mutation near the C terminus (C-terminal sequence: FTD (wild-type)→VH (mutant)) was incubated for 20 min with unilamellar vesicles and visualized by cryoEM and negative-staining EM. The results demonstrate that the VH mutant retains the ability to form pores in lipid bilayers but cannot form filaments. A comparison of the wild-type and mutated C terminus is shown below. d, Quantification of filament formation by 20 μM pro-RegIIIα, wild-type (wt) and C-terminal mutant (VH) RegIIIα in the presence of vesicles. Results are representative of counts from three different areas. nd, not detected. The results show that pro-RegIIIα, which cannot form pores, also cannot assemble into filaments.
We examined the functional properties of the RegIIIα VH mutant carrying a mutation near the C terminus (C-terminal sequence: FTD (wild-type)→VH (mutant)), thus truncating the protein near the C terminus. The VH mutant lacks the ability to form filaments but retains the ability to form pores. In accordance with its pore-forming activity, the RegIIIα VH mutant retained membrane toxicity against liposomes and live bacteria. In fact, membrane toxicity was modestly enhanced in the RegIIIα VH mutant, suggesting that trapping of the pore complexes in filaments inhibits their membrane permeabilizing activity. This function contrasts with that of human α-defensin-6 filaments, which directly trap bacteria in ‘nanonets’20. a, 1.0 μM wild-type (wt) and RegIIIα (VH) mutant was added to 10 μM carboxyfluorescein-loaded liposomes and dye release was monitored. The detergent octylglucoside (OG) was added at the end of the experiment to disrupt remaining liposomes. b, Initial rate of liposome dye release (10 μM lipid) as a function of wild-type and mutant RegIIIα concentration. c, 5.0 μM wild-type or RegIIIα (VH) mutant was assayed for membrane disruptive activity towards whole bacteria using the SYTOX uptake assay described in Fig. 1. Assays were performed in triplicate. Error bars indicate s.d.; ***P < 0.001.
a, Raw image of a single filament. b, c, Comparison of the average power spectrum of cryoEM images of individual short helical segments (b) and the average power spectrum (c) from the projections of the three-dimensional reconstruction at evenly sampled rotation angles around the helical axis. Layer lines 1, 5 and 9 were labelled, and layer line 4 was clearly visible. d, Symmetry variability (Δϕ and Δz) in the cryoEM data set. The reconstruction from the aligned images was imposed with symmetry parameters that vary around the centre pair (Δϕ = 54.5° and Δz = 18.4 Å), and the experimental data set was classified into nine bins by projection matching. The populations in these classes were exhibited in a three-dimensional histoplot. Even though the central bin is the most populated, the distribution is approximately flat. e, Fourier shell correlation (FSC) calculated from the two independent volumes but windowed in different boxes. The strong symmetry in the two volumes led to the FSC ∼0.2 at the Nyquist frequency. The first fast drop of FSC curve to 0.5 was elected to give an approximate estimate of resolution. f, Number of the filament images aligned with each reference projection from the three-dimensional model in the last round of refinement. The projections from the three-dimensional model evenly sampled the orientation space. As expected, the distribution is fairly flat. g–j, Statistical analyses of the RegIIIα filament structure. g, First four eigenimages from the multivariate statistical analysis of the centred filaments in the data set that were padded to 320 pixels in size. The second and third images lack mirror symmetry around the central line, suggesting the parity is odd. The fourth image shows the significant local bending of the filaments, a major limiting factor for us in reaching a better resolution in our reconstruction. h, A good class average after the multivariate statistical analysis and hierarchical classification. i, Square root of calculated power spectrum of the class average in h. The tip of the red arrowhead points at 10.4 Å. j, The layer lines in the average power spectrum of the rotational projections from the final reconstruction without symmetry imposition extend isotropically to ∼9.2 Å (yellow circle), and further along the vertical direction (helical axis). k, l, Docking of the RegIIIα crystal structure into the cryoEM map. k, The three-dimensional reconstruction calculated from the images in the central bin, d, with a hexameric pore highlighted. l, Stereo image showing docking of the RegIIIα crystal structure in the cryoEM density map of one subunit out of the reconstruction.
a, Table showing data collection and refinement statistics for the active RegIIIα crystal structure. b, Crystallographic B-factor map of the active RegIIIα structure showing areas of conformational flexibility. Red indicates greater flexibility.
a, Mutagenesis of Lys 93 (K93) with conservative amino acid substitutions (Arg (R) and His (H)) does not alter membrane toxicity of RegIIIα. 5 μM of wild-type, Lys93Arg mutant, or Lys93His mutant RegIIIα was added to 100 μM carboxyfluorescein-loaded liposomes and dye release was monitored. These mutants retain membrane toxicity, in contrast to Lys93Ala (Fig. 3e), suggesting the importance of positive charges at these sites. b, Filamentation of RegIIIα mutants (Lys93Ala (K93A) and Glu114Gln (E114Q)) correlates with membrane toxicity. 20 μM RegIIIα Lys93Ala (left panel) or Glu114Gln (right panel) was incubated for 20 min with unilamellar vesicles and visualized by negative-staining EM. The results demonstrate that the non-toxic Glu114Gln mutant, unlike the toxic Lys93Ala mutant, assembles into filaments.
a, Top-down view of the numeric grid and complex boundary used in the elasticity calculations to represent the upper leaflet. The protein complex occupies the white space in the centre, and the membrane–protein contact curve is the red–white boundary. The membrane is modelled in all non-white regions. The rectangular grid for the elasticity solver is shown here coloured by the membrane bending energy density (red is high bending energy and blue is low bending energy). This calculation corresponds to the membrane bending shown in Fig. 3g. b–d, Numeric convergence of the model. b, Convergence of the elastostatic energy. In all panels, per cent error was calculated as 100|(E(n) – E(nmax))/E(nmax)|, where E(n) is energy calculated with n grid points, and nmax is maximum number of grid points used. The elastic energy converges smoothly as n increases, and we used n = 400 in both the x and y directions for all calculations in the main text, which gives a 5% error. c, Convergence of the electrostatic energy. Per cent error of the dipole charge–protein interaction energy (diamonds), protein solvation energy (squares), anionic lipid charge–protein interaction energy (circles) and the total electrostatic energy (triangles) are shown as a function of the grid discretization. A value of n = 161 was used for the calculations discussed in the main text resulting in a total electrostatic error of 2.5%. d, Convergence of the non-polar energy. A discretization of n = 100 points was used for the calculations reported in the main text, and this has a very small error on the order of 0.1%. Values used for calculations in the main text are indicated by an asterisk. e, f, Electrostatic potential of the RegIIIα pore complex. e, In-plane view. The Poisson–Boltzmann equation was solved using APBS after embedding the complex in a low dielectric region mimicking the lipid bilayer21. The low dielectric membrane region is deformed corresponding with the lowest energy shape predicted by our physics-based computational model. Positive (blue) isocontours of the electrostatic potential are drawn at +5 kcal mol−1 e−1. f, Out-of-plane view. All details are identical to those in panel a. Both positive (blue) and negative (red) isocontours of the electrostatic potential are drawn at ±5 kcal mol−1 e−1. g, Table showing bilayer material properties used in the modelling calculations. h, Table showing model parameters. References 24–29 are cited in this figure.
a, RegIIIα pore complex model shown from the side. Arg 166 (R166) is located near the water–membrane interface, indicating that it is positioned to interact with the phospholipid head-groups, whereas Arg 39 is predicted to be exposed to aqueous solvent. Membrane boundaries predicted from the computational calculations are indicated. b, 5 μM of wild-type, Arg166Ala mutant, or Arg39Ala mutant RegIIIα was added to 100 μM carboxyfluorescein-loaded liposomes and dye release was monitored. The experimental results are consistent with the position of these residues relative to the membrane interface in the model.
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Mukherjee, S., Zheng, H., Derebe, M. et al. Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature 505, 103–107 (2014). https://doi.org/10.1038/nature12729
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