INTERACTIONS OF FIBRILLAR PROTEINS WITH LIPIDS: A MOLECULAR DOCKING INSIGHT

The aggregation of misfolded proteins into specific ordered aggregates, amyloid fibrils, associated with more than forty human diseases, currently attracts great research attention in biomedical and nanotechnological aspects. These aggregates and their oligomeric intermediates are thought to exert their toxic action predominantly at the level of cell membranes. In addition, membrane lipids were found in many amyloid deposits in vivo suggesting that lipid molecules are able to incorporate into fibril structure affecting their morphology and mechanical properties. However, the biological implications and structural prerequisites of fibril-lipid interactions still remain unclear. In the present study the molecular docking techniques was employed to explore the interactions between the amyloid fibrils and lipids in the model systems containing the fibrillar forms of lysozyme, insulin, Aβ (1-42) peptide and N-terminal (1-83) fragment of apolipoprotein A-I, as a protein component and cholesterol, cardiolipin or phosphatidylcholine as a lipid component. Using the PatchDock web server and BIOVIA Discovery Studio software, the structural peculiarities of fibril-lipid associates were uncovered. The van der Waals and alkyl/π-alkyl interactions were found to prevail in stabilization of all types of fibril-lipid complexes. The analysis of most energetically favorable docking positions revealed a preferable surface location of lipids and partial penetration of acyl chains of cardiolipin and phosphatidylcholine into fibril grooves

Among a variety of conformational and aggregational states of polypeptide chain the states of amyloid fibrils and their oligomeric precursors are currently regarded as the most dangerous, since they are associated with more than forty of human diseases [1,2].The amyloid-related cytotoxicity is thought to be primarily determined by the impairment of cell membrane integrity through either detergent-like mechanism of membrane damage (lipid extraction from the bilayer) [3][4][5][6][7] or membrane permeation for ions via the formation of pore-like structures [8][9][10].Moreover, it is becoming increasingly apparent that lipids are an integral part of most fibrillar deposits formed in vivo or amyloid fibrils grown in vitro in the presence of lipids [11].The analysis of lipids extracted from various deposits of amyloid fibrils by high-performance thin layer chromatography and mass spectrometry revealed that they may contain more than 10 % of lipid (by dry weight), with major lipid constituents such as cholesterol and sphingomyelin, and minor fraction of sulfatides, cholesterol esters and fatty acids [12].In the paired helical filaments of tau protein formed in Alzheimer disease the bound lipids were represented by the galactocerebrosides and phosphatidylcholines [13].It is hypothesized that the presence of lipids as an integral part of many amyloid deposits in vivo, is fundamental for biological activity of fibrillized proteins and is essential for the nucleation, morphology, and mechanical properties of fibrils [11].In the onset of amyloid formation the protein-lipid interactions can favor fibril nucleation, but the lipid uptake is limited to the transfer of a fatty acyl group to serine or lysine residues [14], or transfer of a reactive aldehyde fragment produced by oxidative damage to a lipid [15].Lipids are most likely to associate with amyloid assembly at the stages of protein oligomerization and protofilament formation [1].Despite extensive studies of amyloid cytotoxicity, the mechanisms by which lipids may become incorporated into fibril structure still remain poorly understood.In view of this, the aim of the present study was to elucidate the molecular details of fibril-lipid interactions.The molecular docking technique was employed to explore the complex formation in the systems containing the model fibrils of lysozyme, insulin, Aβ (1-42) peptide and N-terminal (1-83) fragment of apolipoprotein A-I as a protein component and cholesterol, cardiolipin or phosphatidylcholine as a lipid one.

METHODS
To predict the most favorable modes of interactions between the amyloid fibrils and lipids, the molecular docking was performed using the PatchDock algorithm built upon a geometry-based shape complementarity principle.The implementation of this algorithm involves the segmentation of molecular shape into patches of different geometry; matching of these patches and evaluation of the docking positions by geometric shape complementarity fit and atomic desolvation energy scoring function [20].The model amyloid fibrils of hen egg white lysozyme, Aβ and apoliporotein A-I were constructed using the CreateFibril tool based on the translational and rotational affine transformations providing several copies of a certain fragment of fibril core, whose subsequent stacking produces the elongated fibrillar aggregate [21].To this end, the following amino acid sequences from fibril core were used: i) K-peptide of lysozyme, GILQINSRW, residues 54-62 of wild-type protein (LzF); ii) the 24-50 fragment from G26R-mutated apolipoprotein A-I, DSRRDYVSQFEGSALGKQLNLKLLDNW (apoA); iii) the 1-42 fragment from Aβ protein, LVFFAEDVGSNKGAIIGLMVGGVVIA (Aβ).The input structures for CreateFibril were generated from the monomers in the β-strand conformation with PatchDock (LzF, apoA) or were obtained from the Protein Data Bank (Aβ tetramer, PDB code 2BEG).The twisting angle was taken as 15°, the value recovered previously for the most stable conformations of fibrillar LzF, Aβ and apoA.The selected docking poses were visualized with the UCSF Chimera software (version 1.14) and analyzed with BIOVIA Discovery Studio Visualizer, v21.1.0.20298,San Diego: Dassault Systemes; 2021.The prominent binding sites were also predicted through the DoGSiteScorer server (https://proteins.plus/2ozr#dogsite).

RESULTS AND DISCUSSION
While analyzing the predictions of PatchDock tool, the fibril-lipid binding modes were ranked according to the global binding energy (GBE), so that the complexes with the lowest binding energy were selected for deeper evaluation (Fig. 1).Further analysis of the docked structures was focused on the following characteristics: i) the location of lipid binding sites on/within amyloid fibril and interacting amino acid residues; ii) the types of interactions accounting for stabilization of the most energetically favorable amyloid-lipid complexes; iii) the atomic contact energy (ACE).

C
As seen in Table 1, the global energy of lipid-fibril binding falls in the range from -69.25 to -7.26 kcal/mol.It was found that or all examined systems the values of GBE decrease in the row Chol → CL → POPC, indicating that the neutral phospholipid POPC forms the most stable complexes with fibrillar proteins.Remarkably, analogous tendencies were revealed also for atomic contact energies, i.e., the lowest ACE values were observed for POPC, while the highest -for Chol.
For all fibrillar proteins under consideration cholesterol was found to display some penetration into the amyloid grooves with the shallowest location of sterol being observed for the lysozyme fibrils (Fig. 1, A).Furthermore, as follows from the Discovery Studio analysis, all amyloid-cholesterol complexes are stabilized by van der Waals and alkyl/π-alkyl interactions.Specifically, Chol participates in the strong alkyl/π-alkyl interactions with His, Leu, Ile amino acid residues of Aβ amyloid fibrils, Tyr of Ins fibrils and Leu of fibrillar Lz.In turn, van der Waals protein-sterol contacts are ensured by Asn residues of apoA and Ins fibers, and predominantly by Gly and Leu residues of Aβ(1-42) and Lz.Additionally, π-σ contacts between His residues of Aβ(1-42) and Chol favor the intercalation of sterol into the amyloid fibril.As shown in Fig. 1, B cardiolipin are characterized mostly by superficial location in the complexes with amyloid fibrils which can be explained by the steric restrictions arising from the bulky structure of this phospholipid containing four acyl chains.No specific interactions were revealed for the association of Aβ(1-42) and Ins fibers with CL acyl tails, where amyloid-lipid assemblies were found to be stabilized predominantly by van der Waals and alkyl interactions (Fig. 1, B).Furthermore, hydrogen bonds were formed between His of Aβ(1-42) and CL headgroup.In turn, the binding of fibrillar apoA-83 and Lz to anionic cardiolipin exhibited more complex behavior.Specifically, apart from van der Waals, H-bond and alkyl contacts, Asn residues of apoA-83 are involved in the metal-acceptor and unfavorable positive-positive interactions with CL.The complexation of CL with Lz amyloid fibers is stabilized by a set of van der Waals, attractive charge, and unfavorable positive-positive contacts.Interestingly, Trp residues were found to be responsible for all these types of interactions.
Shown in Fig. 1, C are the docking poses of the fibril complexes with POPC which demonstrate the preferable surface location of POPC with some protrusion of its acyl chains into fibril grooves.Similar to Chol and CL, the association of POPC with all types of amyloid fibers studied here was found to be stabilized by van der Waals and alkyl/π-alkyl interactions.Additionally, conventional and carbon hydrogen bonds were formed between Gln, Gly, Leu, Tyr amino acid residues and fibrillar Aβ(1-42), Ins and Lz.Furthermore, attractive charge and metal-acceptor contacts were revealed for apoA and Lz amyloid fibrils, while the covalent bond between Tyr residue of Ins and phosphate group of POPC was formed in the case of Ins fibers.Interestingly, unfavorable positive-positive interactions were found to contribute also into POPC complexation with fibrillar Lz.In the present context, it is noteworthy that unfavorable interactions may appear due to several limitations of molecular docking protocol: i) unaccounted flexibility of protein backbone, ii) insufficient conformational sampling of the protein because of repeated fibrillar structure, iii) steric restrictions.Importantly, most of the interacting amino acid residues defined by the Discovery Studio based on the PatchDock analysis, were found to be within the binding pockets predicted by the DoGSiteScorer server.The DoGSiteScorer is a grid-based method that uses a difference of Gaussian filter to detect potential binding pockets based on three-dimensional structure of the protein and ligand molecules.Specifically, the calculations of the amyloid fibril structures via DoGSiteScorer with respect to every lipid allowed us to identify from 2 to 8 binding pockets.However, for every amyloid-lipid complex the binding pocket with the highest score, calculated through the DoGSiteScorer, almost coincided with the binding site, revealed by the molecular docking.The structural properties of the binding pockets are shown in Table 2.

CONCLUSIONS
In summary, the present study provides additional arguments in favor of the possibility of direct interaction of individual lipid molecules with amyloid fibrils.The model fibril-lipid systems whose protein component was represented by the fibrillar lysozyme, insulin, Aβ (1-42) peptide and N-terminal (1-83) fragment, while a lipid component involved cholesterol, cardiolipin or phosphatidylcholine were investigated using the molecular docking tool.The analysis of the results obtained with PatchDock showed that Asn, Gly, Glu, Leu and Trp are the most common amino acid residues in the formation of van der Waals amyloid-lipid contacts for all types of lipids and proteins studied in the present contribution.In turn, a predominant role of Leu, Tyr and Trp among other residues in providing the alkyl/π-alkyl interactions was demonstrated.Further studies in this direction are important for uncovering the determinants of amyloid biological activity and the development of therapeutics for amyloid diseases.

Table 1 .
Parameters of molecular docking of amyloid fibrils with lipid molecules

Table 2 .
Structural characteristics of binding pockets for lipids within the amyloid fibrils predicted by DoGSiteScorer