GORILLA TO THE RESCUE DETERMINING A NOVEL ANTIMICROBIAL FIBRIL STRUCTURE
The emergence of resistant and aggressive microbial infections calls for novel and effective drugs. Crystal structures of antimicrobial peptides revealed densely packed helical fibrils, while structure-guided mutagenesis supported the role of self-assembly in activity. The findings might facilitate the design of biomaterials with enhanced selectivity, bioavailability, and shelf-life.
Antimicrobial peptides (AMPs) are produced by many organisms as the first line of defence against pathogens and for the modulation of the immune system. They can provide new therapeutic avenues to combat severe infections, kill cancerous cells and treat autoimmune disorders. Moreover, AMPs are thought to induce less resistance compared to conventional antibiotics. Yet, their relatively low efficacy and bioavailability, and lack of chemical stability has discouraged development into therapeutic agents. Fibrillation of antimicrobial peptides into highly stable structures can provide immense stability against heat, shear force, and chemical and proteolytic degradation. This offers new possibilities for therapeutics, and for various biomedical and technological applications.
LL-37 is a mammalian cationic AMP that plays pivotal roles in the innate immune system. LL-3717-29 is a tentative proteolytic cleavage segment of LL-37, showing a different spectrum of antibacterial activity compared to LL-37 and other fragments, and which generates an amphipathic helix with a large hydrophobic moment. This study shows that human and gorilla LL-3717-29 form wide fibrils around bacterial cells (Figure 33). Crystal structures of helical AMPs, and of functional fibrils, are rare. Indeed, human LL-3717-29 was reluctant to crystallise, and crystals formed in only one out of ~1500 conditions tested. The crystals were sensitive to ice formation, and cryo- protectants had to be intensively screened. Finally, X-ray diffraction data were obtained to 1.35 Å resolution, but phases were hard to determine via molecular replacement or direct methods. In contrast, the gorilla LL-3717-29 homologue, having one amino acid substitution (corresponding to human F17S), formed crystals in different conditions. X-ray diffraction data were collected at beamline ID23-2, up to 1.1 Å resolution. This allowed phasing by molecular replacement using a polyalanine idealised helix as the search model. Phases of the human LL-3717-29 were then obtainable via molecular replacement using the refined structure of the gorilla LL3717-29 as the search model.
The human and gorilla crystal structures (PDB IDs: 6S6M and 6S6N, respectively) have
similar space group and unit cell dimensions. The structures revealed densely packed helices forming an elongated hexameric arrangement featuring a nanotube along the fibril axis (Figures 33 and 34). The helices are packed into four-helix bundles with a hydrophobic core, while polar interactions mediate interfaces between bundles. The surface features zig- zagged hydrophobic and positively charged patches, suggesting interactions and disruption of negatively charged lipid bilayers such as bacterial membranes. This overall arrangement is similar between the human and gorilla LL-3717-29, differing only in the two N-terminal positions that line the central pore (Figure 34), with the gorilla structure showing a more occluded pore with extended side chains facing inward. Interestingly, the gorilla peptide displays weaker antibacterial activity, towards both gram positive and negative bacteria, compared to the human homologue.
Fig. 33: A transmission electron micrograph of gorilla LL-3717-29 fibrils assembling on and around an M. luteus bacterium cell. The crystal structure of the fibrils is illustrated on the micrograph, represented as helical ribbons
coloured by hydrophobicity (orange to blue indicating hydrophobic to hydrophilic residues). Credit: Sharon Amlani.