Transmembrane pores formed by human antimicrobial peptide LL-37

Abstract

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23-33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

Figure 1

OCD. (A) Bottom red spectrum is the OCD of an open sample of P/L = 1/50, unchanged from 50% to 100% RH. As excessive water condensed on the sample, the spectrum gradually changed in time from bottom to top (the blue color content of the spectra increased with time), whereas the amount of sample in the CD beam path decreased because the surface of the sample slowly slid downward. It took 40 min of continuous OCD scanning from the red to the blue spectrum. The scan time for each spectrum was ∼4 min, including the resetting time, at 1 nm bandwidth, 0.1 nm/point, and 20 nm/min scan rate. Ten spectra were taken, but for clarity only five are shown. (B) At the appearance of the top blue spectrum in A (rescaled in B), the sample was open to 50% RH and held horizontally for ∼10 min. This made the sample stop flowing, and the spectrum turned to the bottom red curve. The blue and red spectra were obtained from the same amount of sample (blue, I spectrum for helices normal to the bilayers; red, S spectrum for helices parallel to the bilayers). The green spectrum was obtained from a sandwiched sample in a swollen state, fit by a linear combination of the I and S spectra (0.4 I + 0.6 S; purple line), indicating that 40% of the helices were oriented normal to the bilayers.

Figure 2

(A) X-ray lamellar diffraction by θ–2θ scan from an open sample of P/L = 1/50 equilibrating at 98% RH (bottom) and 100% RH (top; with an attenuator below q = 0.19 Å⁻¹). Note that at 100% RH, the peaks were strongly damped by layer undulations (30). (B) X-ray grazing-angle scattering from an open sample of P/L = 1/50 equilibrated at 60% RH (bottom) and (top) in a swollen state (covered by a mylar sheet; also with an attenuator below q = 0.04 Å⁻¹). Note that in the swollen state, the first-order peak has the characteristic power-law line shape as predicted by Caillé's theory (12,14) due to layer undulations in the swollen lamella. (C) Grazing-angle scattering from the swollen lamella (B) recorded on the CCD detector, which was oriented with the z axis vertically up. The beam center was at the baseline. The rectangular diffraction peak image was the shape of the x-ray beam cross section. The intensity profile along the z axis is shown at the top of panel B.

Figure 3

Neutron in-plane scattering of a sandwiched sample of P/L = 1/50 under three conditions: equilibrated at 100% RH D₂O (triangles), equilibrated with excessive D₂O in a swollen state (circles), and equilibrated with excessive H₂O in a swollen state (squares). Inset: Obtained from the circles curve after removing the background (the empty sample cell). The shoulder peak was fit with a Gaussian curve (gray) at 0.085 Å⁻¹.

Figure 4

Analysis of neutron in-plane scattering. (A) P/L = 1/50 (data from Fig. 3, inset, after subtracting the shoulder peak). (B) P/L = 1/100. The dash-dotted curve is ${\left|F\left(q\right)\right|}^2,$ the dashed curve is S(q), and the solid curve is the minimum χ² fit.

Figure 5

X-ray lamellar diffraction of open samples of 1), P/L = 1/50; 2), P/L = 1/80; and 3), pure DOPC (all at ∼98% RH, 25°C). (A) Diffraction patterns. (B) Constructed electron density profiles across one unit cell (the coordinate z normal to the bilayer) from which the PtP was measured and plotted in the inset as a function of P/L.

Figure 6

(A) GUV with red dye in the lipid and green dye in its content was exposed to 5 μM LL-37. Leakage occurred stochastically. 1) t = 0, right before leakage occurred. 2) t = 30 s. 3) t = 60 s. 4) t = 300 s (the GUV was still intact). Within 60 s, the leakage reduced the content dye intensity to ∼10% of the t = 0 value, whereas photobleaching decreased the intensity of a nonleaking GUV to ∼90%. Leakage was complete at t ∼ 200 s. (B) 1) An aspirated GUV was exposed to 0.5 μM LL-37. 2) The protrusion length initially increased, indicating a membrane area expansion without pore formation (the image shows the maximum protrusion). 3) After reaching the maximum, the protrusion length decreased, indicating pore formation in the membrane (see text). In nine runs of aspiration experiments, the average time to reach the maximum protrusion was ∼13 s and the average time to decrease to the original protrusion length (where ΔA/A = 0) was ∼10 s. Both scale bars = 10 μm.