The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17–29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717–29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717–29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.

T he assembly of basic biological molecules into filamentous structures provides ample opportunities to design bioinspired materials for medical and technological applications [1][2][3][4][5][6][7][8] . One such application is addressing the urgent need to fight microbial aggressive, resistance, infections using materials which allow oral bioavailability, stability in harsh conditions, and long shelf-life. Antimicrobial peptides (AMPs) are canonical components of the innate immune system of many organisms 9 . AMP self-assembly bears functional relevance and can enhance antimicrobial activity 10 . Certain AMPs assemble into well-ordered fibrils that resemble amyloids [11][12][13][14] , which are proteins known to form cross-β fibrils composed of tightly mated β-sheets, and have been associated with neurodegenerative and systemic diseases 15,16 . Correspondingly, recent evidence of antimicrobial properties among some human amyloids suggests a potential physiological role of proteins otherwise known as pathological [17][18][19][20][21] .
Investigation of the N-terminal residues which are not buried within the assembly (Supplementary Table 3) but which face the central pore, showed that the F17A and K18A mutants display a similar reduction in antibacterial activity against M. luteus, with a MIC of 60 µM (Fig. 1). Maintaining the positive charge via a K18R substitution, showed a very similar MIC to that of hLL-37 [17][18][19][20][21][22][23][24][25][26][27][28][29] , while the K18H substitution showed slightly reduced activity (Fig. 1). In contrast, substitution to the polar but uncharged glutamine (K18Q) fully abolished activity (Fig. 1). The results suggest that the two residues facing the pore are important for activity, with the positive charge of Lys18 being the critical determinant. We therefore cannot conclude about the specific role of the central pore in activity, as the effect of substitutions might be related to the reduced positive charge 51,52 regardless of its structural location.

Discussion
To conclude, the atomic structures of human and primate antibacterial LL-37 [17][18][19][20][21][22][23][24][25][26][27][28][29] showed a functional supramolecular nanostructure of densely packed amphipathic helices. This assembly into stable fibrils with a surface forming hydrophobic/charged zigzagged belts can be used as scaffolds for wide-ranging applications in bio and nanotechnology, regenerative medicine and bioengineering 53 , with the invaluable advantage of an inherent antibacterial activity. Links between fibril formation and antimicrobial activity are accumulating [11][12][13][14][17][18][19][20][21]54 , and here, we provide atomic-level insight for such example. Further elucidation of the interplay between antimicrobial activity and fibril formation and morphology will aid the design of AMPs with enhanced potency, selectivity, stability, bioavailability, and shelf-life. Successful design of such functional nanostructures with tunable self-assembly might provide novel antibacterial therapeutics or coating of medical devices, and will may target other roles of AMPs in immunomodulation and in killing cancerous cells 9,10,42 .
The LL-37 17-29 structure differs from known helical fibrils such as the toxic cross-α amyloid fibrils of PSMα3, and from structural a c d e f Hydrophilic Hydrophobic Fibril axis 90°3 0°b Fig. 3 The crystal structure of hLL-37 [17][18][19][20][21][22][23][24][25][26][27][28][29] . The crystal structure of hLL-37 [17][18][19][20][21][22][23][24][25][26][27][28][29] was determined at 1.35 Å resolution. The crystal packing shows selfassembly of amphipathic helices into a densely packed, elongated hexameric fibril with a central pore. The fibril is composed of four-helix bundles with a hydrophobic core that associated via a network of polar interaction (Fig. 5). a The assembly is shown as grey ribbons, with two representative four-helix bundles colored green and purple to emphasize orientation in the fibril. fibrils, such as collagen, actin, and fibrinogen. It overall presents a type of self-assembly which, to the best of our knowledge, is distinct from other protein fibrils, with a role in direct killing of bacterial cells still to be fully determined. Despite the different arrangement, the sequence similarity between the human LL-37 and the bacterial PSMα3, and their shared ability to form helical functional fibrils, suggest a possible molecular or structural mimicry mechanism used by the bacteria to provide immuneevasive and survival strategies 55,56 . This also points to potential functional building blocks across kingdoms of life in the form of densely packed amphipathic helical fibrils, complementing the exciting hypotheses about short amyloid peptides serving as prebiotic information-coding molecules [57][58][59] .
Confocal microscopy imaging of bacteria-peptide interaction. M. luteus were grown for 16 h and diluted to an OD 600 = 0.1. FITC-labeled peptides were dissolved in UPddw, sonicated for 3 min, and then added to the bacteria suspension to a final concentration of 30-150 µM (as indicated in the relevant figure); final reaction volume was 100 µl. Control samples contained everything but the peptide or everything but the bacterium. All samples were incubated, in the dark, at 30°C, with shaking at 220 rpm, for 4 h. Thereafter, 1 ml paraformaldehyde 4% (w/v in PBS) was placed over the samples, for 15 min, at room temperature, in the dark. After fixation, samples were washed three times with fresh PBS, and then treated with Hoechst 33342 (10 mg/ml). All samples were applied to µ-Slides VI 0.4 slides (Ibidi, 80666). Confocal images were acquired using an inverted confocal laserscanning microscope LSM 710 (Zeiss) equipped with a C-Apochromat 40× water immersion objective lens (NA 1.2) and a Definite Focus unit in an environmental chamber set at 37°C. The laser wavelengths for excitation were 405 nm (Hoechst) and 488 nm (FITC). Brightfield images were collected from the 405 nm laser. Emission was collected sequentially at 410-497 nm for Hoechst and at 493-797 nm for FITC. The pinhole was set for 1 µm. Image processing was done with the Fiji software.
Cryogenic electron microscopy. Lyophilized human and gorilla LL37 [17][18][19][20][21][22][23][24][25][26][27][28][29] were dissolved in UPddw to 1-5 mM and incubated at 37°C for 3-10 days (as indicated in the relevant figures). Another sample was prepared from 2 mM hLL37 17-29 dissolved in 2.7 mM sodium dodecyl sulfate (SDS) (diluted in UPddw from a 40% stock). Of note, 2.7 mM SDS is at sub critical micelle concentration (CMC). Within a temperature-controlled chamber at 100% relative humidity, 3 µl of each sample were applied to a perforated carbon film supported by an electron microscope grid, which were pre-discharged, as described above. After 3 s, the drop was blotted by Whatman filter paper and liquids were vitrified through rapid plunging of the grids into liquid ethane at its freezing point 64 . Specimens were examined under a FEI Talos 200 C high-resolution electron microscope, at an accelerating voltage of 200 kV, using a Gatan 626 cryo-holder. To minimize electron beam radiation damage, the low-dose imaging mode was used. Images were collected digitally by a FEI Falcon III direct-imaging camera and the TIA software, with the help of the "phase plates" (FEI), to enhance image contrast 65,66 .
Zeta potential measurements. Lyophilized LL37 [17][18][19][20][21][22][23][24][25][26][27][28][29] and the I24A mutant were dissolved in UPddw and incubated at 0.01, 0.1, and 1 mM concentrations at 37°C for 24 h. Electrophoretic mobility measurements were performed in 25°C using a Malvern's Zetasizer Ultra device while samples were measured in folded capillary zeta cell zeta cuvettes (Malvern, DTS1070). Zeta potential was deduced using the Smoluchowski approximation 47 . The cells and the electrodes were washed with ddw three times before and after each sample. Device was equilibrated for 120 s before each sample and then three consecutive measurements were performed. The presented data is the mean of three consecutive measurements and standard deviation indicates the route mean square of the three measurements.
Sequence alignments and proteolytic digestion prediction. Sequence alignment between LL37 17-29 and PSMα3 was performed using the MAFFT server 72 . Amino acids are colored by their physicochemical properties 73 . Proteolytic digestion sites in the human LL37 were predicted using the Expasy's PeptideCutter Tool 74 .
Calculations of structural properties. The electrostatic potential map, hydrophobicity and B factor scales presented in the figures were created using Chimera 71 . The values of the hydrophobicity scale were according to Kyte and Doolittle 75 . The electrostatic potential was calculated using APBS-PDB2PQR 76 . Helix amphipathicity and the hydrophobic moment (Supplementary Table 1) were calculated with HeliQuest 77 .
Solvent-accessible surface area calculations. Solvent-accessible surface areas (SASAs) were calculated using AREAIMOL, with a probe radius of 1.4Å7 8,79 , via the CCP4 package 69 . The solvent-accessible buried surface area of each chain in the asymmetric unit was calculated as the area difference between the isolated chain and the chain within the fibril assembly, and is presented as the percentage of the total SASA of the chain. The SASA per residue within different isolated helical assemblies are presented in Supplementary Table 3.