Anna P. Tolstova

The review deals with the application of Molecular Dynamics (MD) to the structure modeling of beta-amyloids (Aβ), currently classified as intrinsically disordered proteins (IDPs). In this review, we strive to relate the main advances in this area but specifically focus on the approaches and methodology. All relevant papers on the Aβ modeling are cited in the Tables in Supplementary Data, including a concise description of the applied approaches, sorted according to the types of the studied systems: modeling of the monomeric Aβ and Aβ aggregates. Similar sections focused according to the type of modeled object are present in the review. In the final part of the review, novel methods of general IDP modeling not confined to Aβ are described.

Src kinase is one of the key regulators of cellular metabolism and is dysregulated in numerous diseases, including cancer, neurodegenerative diseases, and particularly Alzheimer’s disease. Despite its therapeutic importance, its full-length structure has never been obtained before, as it contains an intrinsically disordered regulatory region, SH4UD. The SH4UD region is crucial for Src activation, functional dimerization, and regulation by other kinases. In this study, we used the replica exchange molecular dynamics approach with a hybrid temperature and Hamiltonian tempering to obtain the conformational ensemble of full-length Src kinase in its non-phosphorylated state and in the presence of its two key regulatory phosphorylations: pY419 and pY530. The representative structures and simulation trajectories of non-phosphorylated pY419 and pY530 Src are available in open access. We demonstrate that pY419 phosphorylation, which is associated with Src activation, enhances its motility, whereas inhibited pY530 Src preserves relatively compact conformation. This study also provides insights into how SH4UD contributes to Src substrate binding, dimerization, and autophosphorylation, highlighting the putative role of 14-RRR-16 in this process.

Beta amyloid peptide Aβ 1-42 (Aβ42) has a unique dual role in the human organism, as both the peptide with an important physiological function and one of the most toxic biological compounds provoking Alzheimer's disease (AD). There are several known Aβ42 isoforms that we discuss here that are highly neurotoxic and lead to the early onset of AD. Aβ42 is an intrinsically disordered protein with no experimentally solved structure under physiological conditions. The objective of this research was to establish the appropriate molecular dynamics (MD) methodology and model a uniform set of structures for the Aβ42 isoforms that form the core of this study. For that purpose, force field selection and verification including convergence testing for MD simulations was made. Replica exchange MD and conventional MD modeling of several Aβ42 and Aβ16 isoforms that have neurotoxic and amyloidogenic effects impacting the severity of Alzheimer's disease were carried out with the optimal force field and solvent parameters. A standardized ensemble of structures for the Aβ42 and Aβ16 isoforms covering 30-50% of the conformational ensembles extracted from the free energy minima was calculated from MD trajectories. The resulting data set of modeled structures includes Aβ42 wild type, isoD7, pS8, D7H, and H6R-Aβ42 and Aβ16 wild type, isoD7, pS8, D7H, and H6R-Aβ16. The representative structures are given in the Supporting Information; they are open for public access. In the study, we also evaluated the differences between the structures of Aβ42 isoforms and speculate on their possible relevance to the known functions. Utilizing several representative structures for a single disordered protein for docking, with their subsequent averaging by conformations, would markedly increase the reliability of docking results.

Amyloid-β (Aβ) is a peptide formed by 39–43 amino acids, heterogenous by the length of its C-terminus. Aβ constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer’s disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aβ seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aβ isoforms, among which the most common is Aβ with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aβ aging. Aβ molecules with isoD7 (isoD7-Aβ) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aβ. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aβ is based on the rigidly structured segment 11-EVHH-14, located in the Aβ metal binding domain (Aβ₁₆). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aβ with Taiwan (D7H) mutation (D7H-Aβ). In this study, the D7H-Aβ metal binding domain (D7H-Aβ₁₆) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aβ oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aβ, as well as isoD7-Aβ in combination with two Aβ molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aβ species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Beta-amyloid peptide (Aβ) is a ligand associated with RAGE (Advanced glycosylation end product-specific receptor). Aβ is translocated in complexes with RAGE from the blood to brain across the blood–brain barrier (BBB) by transcytosis. Aβ and its isoforms are important factors in the Alzheimer’s disease (AD) pathogenesis. However, interaction with RAGE was previously studied for Aβ but not for its isoforms. The present study has been directed at identifying the key interaction interfaces between RAGE and Aβ isoforms (Aβ₄₀, Aβ₄₂, phosphorylated and isomerized isoforms pS8-Aβ₄₂, isoD7-Aβ₄₂). Two interfaces have been identified by docking: they are represented by an extended area at the junction of RAGE domains V and C1 and a smaller area linking C1 and C2 domains. Molecular dynamics (MD) simulations have shown that all Aβ isoforms form stable and tightly bound complexes. This indicates that all Aβ isoforms potentially can be transported through the cell as part of a complex with RAGE. Modeling of RAGE interaction interfaces with Aβ indicates which chemical compounds can potentially be capable of blocking this interaction, and impair the associated pathogenic cascades. The ability of three RAGE inhibitors (RAP, FPS-ZM1 and RP-1) to disrupt the RAGE:Aβ interaction has been probed by docking and subsequently the complexes’ stability verified by MD. The RP-1 and Aβ interaction areas coincide and therefore this inhibitor is very promising for the RAGE:Aβ interaction inhibition.

Beta-amyloid (Aβ) has a dual role, both as an important factor in the pathology of Alzheimer’s disease and as a regulator in brain physiology. The inhibitory effect of Aβ₄₂ oligomers on Na,K-ATPase contributes to neuronal dysfunction in Alzheimer’s disease. Still, the physiological role of the monomeric form of Aβ₄₂ interaction with Na,K-ATPase remains unclear. We report that Na,K-ATPase serves as a receptor for Aβ₄₂ monomer, triggering Src kinase activation. The co-localization of Aβ₄₂ with α1- and β1-subunits of Na,K-ATPase, and Na,K-ATPase with Src kinase in SH-SY5Y neuroblastoma cells, was observed. Treatment of cells with 100 nM Aβ₄₂ causes Src kinase activation, but does not alter Na,K-ATPase transport activity. The interaction of Aβ₄₂ with α1β1 Na,K-ATPase isozyme leads to activation of Src kinase associated with the enzyme. Notably, prevention of Na,K-ATPase:Src kinase interaction by a specific inhibitor pNaKtide disrupts the Aβ-induced Src kinase activation. Stimulatory effect of Aβ₄₂ on Src kinase was lost under hypoxic conditions, which was similar to the effect of specific Na,K-ATPase ligands, the cardiotonic steroids. Our findings identify Na,K-ATPase as a Aβ₄₂ receptor, thus opening a prospect on exploring the physiological and pathological Src kinase activation caused by Aβ₄₂ in the nervous system.

Alzheimer’s disease (AD) is a neurodegenerative disease accompanied by progressive cognitive and memory dysfunction due to disruption of normal electrotonic properties of neurons and neuronal loss. The Na,K-ATPase interaction with beta amyloid (Aβ) plays an important role in AD pathogenesis. It has been shown that Na,K-ATPase activity in the AD brain was significantly lower than those in age-matched control brain. The interaction of Aβ₄₂ with Na,K-ATPase and subsequent oligomerization leads to inhibition of the enzyme activity. In this study interaction interfaces between three common Aβ₄₂ isoforms, and different conformations of human Na,K-ATPase (α1β1) have been obtained using molecular modeling, including docking and molecular dynamics (MD). Interaction sites of Na,K-ATPase with Aβ₄₂ are localized between extracellular parts of α- and β- subunits and are practically identical for Na,K-ATPase at different conformations. Thermodynamic parameters for the formation of Na,K-ATPase:Aβ₄₂ complex at different conformations acquired by isothermal titration calorimetry (ITC) are similar, which is in line with the data of molecular modeling. Similarity of Na,K-ATPase interaction interfaces with Aβ in all conformations allowed us to cross-screen potential inhibitors for this interaction and find pharmaceutical compounds that could block it.

Due to its unique properties and high biomedical relevance fibrinogen is a promising protein for the development of various matrixes and scaffolds for biotechnological applications. Fibrinogen molecules may form extensive clots either upon specific cleavage by thrombin or in thrombin‐free environment, for example, in the presence of different salts. Here, we report the novel type of non‐conventional fibrinogen clot formation, which is mediated by myeloperoxidase and takes place even at low fibrinogen concentrations (<0.1 mg/ml). We have revealed fibrillar nature of myeloperoxidase‐mediated fibrinogen clots, which differ morphologically from fibrin clots. We have shown that fibrinogen clotting is mediated by direct interaction of myeloperoxidase molecules with the outer globular regions of fibrinogen molecules followed by fibrinogen unfolding from its natural trinodular to a fibrillar structure. We have demonstrated a major role of the Debye screening effect in regulating of myeloperoxidase‐induced fibrinogen clotting, which is facilitated by small ionic strength. While fibrinogen in an aqueous solution with myeloperoxidase undergoes changes, the enzymatic activity of myeloperoxidase is not inhibited in excess of fibrinogen. The obtained results open new insights into fibrinogen clotting, give new possibilities for the development of fibrinogen‐based functional biomaterials, and provide the novel concepts of protein unfolding.

It is widely accepted that the addition of zinc leads to the formation of neurotoxic nonfibrillar aggregates of beta-amyloid peptides Aβ₄₀ and Aβ₄₂ and at the same time destabilizes amyloid fibrils. However, the mechanism of the effect of zinc on beta-amyloid is not fully understood. In this study, a fast zinc-induced aggregation of Aβ₁₆ (as compared to a system without zinc) via the formation of Aβ₁₆ dimers with one zinc ion coordinated in the metal-binding site ₁₁EVHH₁₄, followed by their polymerization, has been studied by molecular dynamics. The best aggregation was shown by the system composed of Aβ₁₆ dimers bound by one zinc ion, with no additional zinc in solution. The presence of Aβ₁₆ dimers was a major condition, sufficient for fast aggregation into larger complexes. It has been shown that the addition of zinc to a system with already formed dimers does not substantially affect the characteristics and rate of aggregation. At the same time, an excessive concentration of zinc at the early stages of the formation of conglomerates can negatively affect aggregation, since in systems where zinc ions occupied the ₁₁EVHH₁₄ coordination center and the His6 residue of every Aβ₁₆ monomer, the aggregation proceeded more slowly and the resulting complexes were not as large as in the zinc-free Aβ system. Thus, this study has shown that the formation of Aβ₁₆ dimers bound through zinc ions at the ₁₁EVHH₁₄ sites of the peptides plays an important role in the formation of neurotoxic non-fibrillar aggregates of beta-amyloid peptide Aβ₁₆. The best energetically favorable structure has been obtained for the complex of two Aβ₁₆ dimers with two zinc ions.