Helical antimicrobial peptides assemble into protofibril scaffolds that present ordered dsDNA to TLR9

Abstract

Amphiphilicity in ɑ-helical antimicrobial peptides (AMPs) is recognized as a signature of potential membrane activity. Some AMPs are also strongly immunomodulatory: LL37-DNA complexes potently amplify Toll-like receptor 9 (TLR9) activation in immune cells and exacerbate autoimmune diseases. The rules governing this proinflammatory activity of AMPs are unknown. Here we examine the supramolecular structures formed between DNA and three prototypical AMPs using small angle X-ray scattering and molecular modeling. We correlate these structures to their ability to activate TLR9 and show that a key criterion is the AMP's ability to assemble into superhelical protofibril scaffolds. These structures enforce spatially-periodic DNA organization in nanocrystalline immunocomplexes that trigger strong recognition by TLR9, which is conventionally known to bind single DNA ligands. We demonstrate that we can "knock in" this ability for TLR9 amplification in membrane-active AMP mutants, which suggests the existence of tradeoffs between membrane permeating activity and immunomodulatory activity in AMP sequences.

Fig. 1

Small-angle X-ray scattering (SAXS) structure of melittin-double-stranded DNA (dsDNA) complexes. a Representative SAXS data of an isoelectric melittin-dsDNA complex with the first Bragg peak q₁₀₀ indicated. b The magnified data region shows details of diffraction features q_hkl, which index well to a three-dimensional tetragonal lattice. c To obtain the lattice parameters, we fit the measured Bragg peak positions to predicted reflections using multiple linear regression. The lattice parameters of the melittin-dsDNA complex are d = 3.64 nm and c = 3.28 nm (R² = 0.999). d Structure of the melittin monomer (PDB: 2MLT_A). Cationic residues are colored blue, hydrophobic residues are colored white, and polar uncharged residues are colored green. e Schematic of parallel dsDNA rods within the melittin-dsDNA tetragonal lattice labeled with lattice parameters obtained from SAXS. The average domain size of the melittin-dsDNA lattice is L ~ 62.4 nm

Fig. 2

Structure of melittin-double-stranded DNA (dsDNA) complexes from molecular simulations. a Side and top views of the melittin protofibril self-assembled in the presence of dsDNA. The cross-sectional dimensions of the protofibril is 2.7 nm × 2.7 nm. The helical pitch of the protofibril is 3.28 nm per turn. Top (b) and side (c) views of an 8 × 8 melittin-dsDNA square lattice from molecular dynamics simulations. Columnar dsDNA is colored red, and the melittin protofibril is colored green (N to C terminus polarity) and teal (C to N terminus polarity). Random polarity of the melittin protofibril is assumed. The inter-dsDNA spacing from the molecular model obtained from simulation is d = 3.64 nm, which agrees well with the predicted inter-dsDNA spacing from small-angle X-ray scattering (SAXS). The out-of-plane lattice parameter of c = 3.28 nm also agrees with the simulated pitch of the melittin protofibril. d There is good agreement between the observed structure factor peaks from SAXS with peaks recovered from theoretical X-ray scattering of the lattice model in b and c from CRYSOL

Fig. 3

Structure of LL37-double-stranded DNA (dsDNA) complexes from small-angle X-ray scattering (SAXS) and molecular simulations. a Representative SAXS data from an isoelectric LL37-dsDNA complex. All observed reflections are labeled with their respective Miller indices (hk). b Two-dimensional square lattice reflections (q_hk) from a are fitted to $q_{hk}=\frac{2\pi }{d}\sqrt{h^2+k^2}$, yielding a lattice parameter of d = 3.40 nm (R² = 0.999). c Structure of the LL37 monomer (PDB: 2K6O). Cationic residues are colored in blue and hydrophobic residues are colored in white. d Schematic of the LL37-dsDNA square columnar lattice with an inter-dsDNA spacing of d = 3.40 nm. The average domain size of the LL37-dsDNA lattice is L ~ 16.1 nm. e Side and top views of the self-assembled LL37 protofibril structure formed in the presence of dsDNA. The protofibril has rough cross-sectional dimensions of 2.5 nm × 2.5 nm. The helical pitch of the protofibril is 6.8 nm per turn. f Top view of a 3 × 3 LL37-dsDNA square lattice from molecular dynamics simulations. Columnar dsDNA is colored red, and the LL37 protofibril is colored green (N to C terminus polarity) and teal (C to N terminus polarity). Random polarity of the LL37 protofibril is assumed. The inter-dsDNA spacing from the molecular model is 3.4 nm, which agrees with the inter-dsDNA spacing predicted from SAXS. g There is good agreement between the observed structure factor peaks from SAXS with peaks recovered from theoretical X-ray scattering of the lattice model in f from CRYSOL

Fig. 4

Structure of buforin-double-stranded DNA (dsDNA) complexes. a Representative small-angle X-ray scattering (SAXS) data of an isoelectric buforin-dsDNA complex. One main diffraction peak is observed at q₁ = 0.170 Å⁻¹ with weak broad features at higher q, consistent with a disordered columnar structure with short-ranged order. b Structure of the buforin monomer (taken from the H2A histone, PDB: 4KGC). See Supplementary Discussion for details regarding the relationship between buforin and H2A. c Illustration of the buforin-dsDNA columnar lattice with average inter-dsDNA spacing of d = 3.70 nm

Fig. 5

LL37-double-stranded DNA (dsDNA) and buforin-dsDNA complexes induce type 1 interferon (IFN) production in a TLR9-dependent manner in mouse macrophages and human plasmacytoid dendritic cells (pDCs). a To measure the ability of AMP-dsDNA complexes to activate TLR9, complexes were incubated with wild-type (WT) and TLR9-knockout (TLR9KO) murine macrophages. IFN-β production was measured and quantified from supernatants using a Bio-ELISA. Mouse genomic dsDNA was used to generate the complexes. Data show TLR9-dependent activation of WT macrophages by LL37-dsDNA and buforin-dsDNA complexes, with minimal to no activation with peptides or dsDNA alone. As a positive control, mouse-specific TLR9 agonist ODN 1826 was used. Both WT and TLR9KO cells responded to the TLR4 agonist lipopolysaccharide (LPS) (n = 3, *p < 0.05, **p < 0.01). b Similarly, AMP-dsDNA complexes were incubated with human pDCs in the absence or presence of chloroquine (1 µM), and IFN-ɑ production was quantified with enzyme-linked immunosorbent assay (ELISA). Data show that pDCs potently release IFN-ɑ in a TLR9-dependent manner in response to LL37-dsDNA and buforin-dsDNA complexes, and this response is attenuated in the presence of chloroquine, which inhibits endosomal acidification and TLR specific signaling (n = 2, *p < 0.05). Error bars denote s.e.m. Group comparisons were calculated using pairwise two-tailed t-tests

Fig. 6

Systematic reduction of the membrane activity of melittin in peptides with conserved double-stranded DNA (dsDNA)-binding activity corresponds with a recovery of TLR9-activating ability. a Small-angle X-ray scattering (SAXS) spectra from PS/PE = 20/80 model membranes incubated with melittin, AR23, RV23, and MM1 at P/L = 1/2. Melittin, AR23, and RV23 induce Pn3m cubic phases in model membranes, while MM1 does not induce appreciable changes compared to controls (Supplementary Figure 7). Correlation peaks corresponding to assigned reflections for the Pn3m cubic phases are labeled for melittin, AR23, and RV23. b SAXS spectra corresponding to the structures of melittin-, AR23-, RV23-, and MM1-DNA complexes. The melittin-DNA SAXS data are reproduced here from Fig. 1 for ease of comparison. Higher-order reflections for the AR23 and RV23 square lattices are labeled. AR23 and RV23 form square columnar lattices with dsDNA (similar to melittin and LL37), while MM1 forms a disordered columnar arrangement. c The absolute value of average negative Gaussian curvature (NGC) is calculated from the lattice parameter (a) of the cubic phases measured in a. Compared to melittin, AR23 and RV23 induce decreasing amounts of NGC in membranes, while MM1 does not induce NGC at all. d Measured TLR9-specific interferon (IFN)-ɑ production (pg mL⁻¹) from human pDCs induced by AMP-dsDNA complexes. Systematic reduction of NGC-inducing ability in melittin-related peptides AR23 and RV23 (b) correlate with a recovery in ability to induce IFN-ɑ from pDCs. MM1 is unable to induce NGC or IFN-ɑ production from pDCs. Error bars denote s.e.m.