Enhanced anti-microbial peptide variant forms novel fibril type

Enhanced anti-microbial peptide variant forms novel fibril type

Protein self-assembly into polymers and fibrils serves different purposes in nature. Amyloid fibrils are a prominent example of protein self-assembly into highly ordered structures, due to their association to neurodegenerative diseases. Recent studies have shown a strong link between anti-microbial peptides (AMPs) and amyloid forming proteins. Anti-microbial activity has been demonstrated for several, well-known amyloids1–7 and many AMPs have been found to form amyloid fibrils under certain conditions. Amyloid fibrils have a characteristic cross-β sheet motif8. However, a variety of different protein fibril structures, including cross α-helical amyloid-like fibrils9–11 and functional α-helical assemblies12 have recently been discovered. In addition to the naturally occurring protein self-assemblies, the design of self-assembling peptides for functional materials has shown great potential13–18. Tuneable features such as pH as a stimulus for self-assembly19–22, have been introduced to overcome challenges in immunogenicity, off-target effects and serum instability and ensure their controlled release23–25.

 In this study we characterised the engineered AMP plectasin variant PPI42 and its self-assembly into helical non-amyloid fibrils. PPI42 contains three point mutations (D9S; Q14K; V36L) and was engineered to improve plectasin’s anti-microbial activity. In our previous study we showed that PPI42’s solution behaviour differs significantly from the wildtype26. PPI42 forms a hydrogel at neutral and basic pH, while the wildtype remains in solution. Using atomic force microscopy (AFM) and negative stain electron microscopy we could show that the hydrogel consists of fibrillar superstructures that are formed by two protofibrils that are arranged in a helical manner (Fig. 1a). By applying various biophysical methods, we attempted to characterise the fibril structure and the onset and reversibility of the fibril formation. We could show that the onset of fibril formation as well as the stoichiometry between fibril and monomers is strongly pH and protein concentration dependent (Fig. 1b). Fibril formation followed sigmoidal kinetics and was accelerated by adding fibril seeds. PPI42 self-assembly into fibrils proved to be completely reversible upon lowering the pH (Fig. 1c). Following the chemical shift of all amino acids as a function of pH, the titratable sidechain of histidine 18 seemed to act as a switch for PPI42’s self-assembly. Circular dichroism measurements and X-ray fiber diffraction showed that the peptide structure of PPI42 differed from the structure of amyloid fibril and PPI42 fibrils proved to be negative for Thioflavin T fluorescence. The secondary structure showed only minor changes upon fibril formation with 𝛼-helix and 𝛽-sheet remaining intact.

Figure 1: a: Images of the fibrils using negative stain EM. PPI42 formed protein fibrils consisting of two coiling protofilaments. b: Fibril formation kinetics for PPI42 fibrils. The fraction of monomer was determined by integration of 1H signals for the I22 and L36 methyl groups relative to time 0. c: left: Light scattering and monitored pH of PPI42 diluted into phosphate buffer pH 7 and subsequently titrated with 1M HCl. The grey line marks the starting point of the titration. Insert shows a zoom in between 0 and 1 hour to visualise the increase in light scattering after dilution into pH 7, while the pH stays constant.  Pictures show the sample at equilibrium after dilution into pH 7 (formed a gel) and after the titration with HCl (liquid). right: Monomeric state of PPI42 before dilution (A), at equilibrium at pH 7 (B) and after titration (C) was assessed with NMR proving that the fibril formation was reversible (signal recovery). (D) shows the wiltype plectasin signal. 

Our biophysical characterisation pointed towards only minor changes in the structure of PPI42 upon fibril formation. However, proving this required a high-resolution structure of the PPI42 fibrils. We were able to determine the cryo-EM structure of the PPI42 fibrils at an overall resolution of 1.97 Å, which allowed unambiguous structure determination and is one of the highest resolutions achieved for fibrils in cryo-EM so far (Fig. 2a). This confirmed that PPI42 remained in a native-like confirmation within the fibril, which resembles the crystal structure of the wildtype closely (Fig. 2b). The asymmetric unit (au) within the fibril consists of seven PPI42 monomers that are related to each other by an average twist of 156.48° and an average axial rise of 3.75 Å. The fibrils are stabilised by a ring-like hydrophobic cluster on the fibril surface and a hydrophobic fibril core (Fig 2c) as well as polar interactions, which are conserved in the protein crystal of the wildtype plectasin. A detailed analysis of PPI42 structure within the fibril showed, that the histidine 18 (H18) sidechain, which is also included in the outer hydrophobic cluster is coordinated differently between PPI42 and the wildtype plectasin due to the Q14K mutation (Fig 2b). This further validated the important role of this sidechain for the fibril formation. Anti-microbial assays confirmed improved potency of PPI42 against Staphylococcicompared to the wildtype plectasin, while maintaining similar potency against Streptococci. We could verify that its anti-microbial activity remains upon release from fibrils, while the fibrils itself are most likely inert based on the position of the amino acids involved in the binding of the bacterial cell wall. However, the PPI42 fibrils might serve as a reservoir for controlled release of the active anti-microbial peptide.

Figure 2: a: High-resolution cryo-EM structure of the protein fibril formed by PPI42. Isosurface representation of the cryo-EM map is shown along the transverse axis and along the longitudinal axis of the fibril (90º rotation). The structure is composed of two coiling protofilaments (p1 and p2). The asymmetric units (au) are related by helical symmetry (axial rise of 25.20 Å and azimuthal angle of 15.75°shown for the p2 protofilament) and coloured differently (green and salmon). Each asymmetric unit is composed of seven monomers. The scale bar is equivalent to 30 Å. b: left: Comparison of the plectasin wildtype crystal structure (black) and PPI42 fibril structure (light blue). The three mutated amino acids are highlighted as sticks. right: Differences in the coordination of H18 between the wildtype and PPI42. The wildtype showed two distinct orientations of H18 while PPI42 only showed one orientation due to Q14K mutation. c: left: Atomic model of one asymmetric unit (au_3) viewed from the side. Each pair of monomers within an asymmetric unit are related to each other by a pseudo helical symmetry (average axial rise of 3.75 ± 0.15 Å and an average twist of 156.48 ± 2.25°).middle: Hydrophobic (red amino acids shown as sticks) interactions in the fibril centre shown in one asymmetric unit with view from the top. Numbering corresponds to the relative monomer position in the asymmetric unit. right: Hydrophobic interactions (black amino acids shown as sticks) forming the outer hydrophobic ring of one protofilament shown in one asymmetric unit with view from the top.

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