Conformational Changes and Unfolding of ß-Amyloid Substrates in the Active Site of γ-Secretase.

Alzheimer's disease (AD) is the leading cause of dementia and is characterized by a presence of amyloid plaques, composed mostly of the amyloid-ß (Aß) peptides, in the brains of AD patients. The peptides are generated from the amyloid precursor protein (APP), which undergoes a sequence of cleavages, referred as trimming, performed by γ-secretase. Here, we investigated conformational changes in a series of ß-amyloid substrates (from less and more amyloidogenic pathways) in the active site of presenilin-1, the catalytic subunit of γ-secretase. The substrates are trimmed every three residues, finally leading to Aß40 and Aß42, which are the major components of amyloid plaques. To study conformational changes, we employed all-atom molecular dynamics simulations, while for unfolding, we used steered molecular dynamics simulations in an implicit membrane-water environment to accelerate changes. We have found substantial differences in the flexibility of extended C-terminal parts between more and less amyloidogenic pathway substrates. We also propose that the positively charged residues of presenilin-1 may facilitate the stretching and unfolding of substrates. The calculated forces and work/energy of pulling were exceptionally high for Aß40, indicating why trimming of this substrate is so infrequent.

GS-SMD server for steered molecular dynamics of peptide substrates in the active site of the γ-secretase complex.

Despite recent advances in research, the mechanism of Alzheimer's disease is not fully understood yet. Understanding the process of cleavage and then trimming of peptide substrates, can help selectively block γ-secretase (GS) to stop overproduction of the amyloidogenic products. Our GS-SMD server (https://gs-smd.biomodellab.eu/) allows cleaving and unfolding of all currently known GS substrates (more than 170 peptide substrates). The substrate structure is obtained by threading of the substrate sequence into the known structure of GS complex. The simulations are performed in an implicit water-membrane environment so they are performed rather quickly, 2-6 h per job, depending on the mode of calculations (part of GS complex or the whole structure). It is also possible to introduce mutations to the substrate and GS and pull any part of the substrate in any direction using the steered molecular dynamics (SMD) simulations with constant velocity. The obtained trajectories are visualized and analyzed in the interactive way. One can also compare multiple simulations using the interaction frequency analysis. GS-SMD server can be useful for revealing mechanisms of substrate unfolding and role of mutations in this process.

Specificities of Protein Homology Modeling for Allosteric Drug Design.

The allosteric binding sites are usually located in the flexible areas of proteins, which are hardly visible in the crystal structures. However, there are notable exceptions like allosteric sites in receptors in class B and C of GPCRs, which are located within a well-defined bundle of transmembrane helices. Class B and C evolved from class A and even after swapping of orthosteric and allosteric sites the central binding site persisted and it can be used for easy design of allosteric drugs. However, studying the ligand binding to the allosteric sites in the most populated class A of GPCRs is still a challenge, since they are located mostly in unresolved parts of the receptor's structure, and especially N-terminus. This chapter provides an example of cannabinoid CB1 receptor N-terminal homology modeling, ligand-guided modeling of the allosteric site in GABA receptor, as well as C-linker modeling in the potassium ion channels where the allosteric phospholipid ligand PIP2 is bound.

The Role of Cholesterol in Amyloidogenic Substrate Binding to the γ-Secretase Complex.

Alzheimer's disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid ß (Aß) plaques in the brain. The γ-secretase complex, which produces Aß, is an intramembrane-cleaving protease consisting of four membrane proteins. In this paper we investigated the amyloidogenic fragments of amyloid precursor protein (substrates Aß43 and Aß45, leading to less amyloidogenic Aß40 and more amyloidogenic Aß42, respectively) docked to the binding site of presenilin, the catalytic subunit of γ-secretase. In total, we performed 9 µs of all-atom molecular dynamics simulations of the whole γ-secretase complex with both substrates in low (10%) and high (50%) concentrations of cholesterol in the membrane. We found that, at the high cholesterol level, the Aß45 helix was statistically more flexible in the binding site of presenilin than Aß43. An increase in the cholesterol concentration was also correlated with a higher flexibility of the Aß45 helix, which suggests incompatibility between Aß45 and the binding site of presenilin potentiated by a high cholesterol level. However, at the C-terminal part of Aß45, the active site of presenilin was more compact in the case of a high cholesterol level, which could promote processing of this substrate. We also performed detailed mapping of the cholesterol binding sites at low and high cholesterol concentrations, which were independent of the typical cholesterol binding motifs.

GPCRsignal: webserver for analysis of the interface between G-protein-coupled receptors and their effector proteins by dynamics and mutations.

GPCRsignal (https://gpcrsignal.biomodellab.eu/) is a webserver devoted to signaling complexes of G-protein-coupled receptors (GPCRs). The recent improvement in cryo-electron microscopy resulted in the determination of a large number of high-resolution structures of GPCRs bound to their effector proteins: G proteins or arrestins. Analyzing the interfaces between receptor and an effector protein is of high importance since a selection of proper G protein or specific conformation of arrestin leads to changes of signaling that can significantly affect action of drugs. GPCRsignal provides a possibility of running molecular dynamics simulations of all currently available GPCR-effector protein complexes for curated structures: wild-type, with crystal/cryo-EM mutations, or with mutations introduced by the user. The simulations are performed in an implicit water-membrane environment, so they are rather fast. User can run several simulations to obtain statistically valid results. The simulations can be analyzed separately using dynamic FlarePlots for particular types of interactions. One can also compare groups of simulations in Interaction frequency analysis as HeatMaps and also in interaction frequency difference analysis as sticks, linking the interacting residues, of different color and size proportional to differences in contact frequencies.

Allosteric Modulation of the CB1 Cannabinoid Receptor by Cannabidiol-A Molecular Modeling Study of the N-Terminal Domain and the Allosteric-Orthosteric Coupling.

The CB1 cannabinoid receptor (CB1R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB1R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB1R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ9-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB1R with ligands: (i) CBD together with THC, and (ii) THC-only. Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB1R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ9-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB1R as well as for the allosteric-orthosteric coupling mechanism.

The Hydrophobic Ligands Entry and Exit from the GPCR Binding Site-SMD and SuMD Simulations.

Most G protein-coupled receptors that bind the hydrophobic ligands (lipid receptors and steroid receptors) belong to the most populated class A (rhodopsin-like) of these receptors. Typical examples of lipid receptors are: rhodopsin, cannabinoid (CB), sphingosine-1-phosphate (S1P) and lysophosphatidic (LPA) receptors. The hydrophobic ligands access the receptor binding site from the lipid bilayer not only because of their low solubility in water but also because of a large N-terminal domain plug preventing access to the orthosteric binding site from the extracellular milieu. In order to identify the most probable ligand exit pathway from lipid receptors CB1, S1P1 and LPA1 orthosteric binding sites we performed at least three repeats of steered molecular dynamics simulations in which ligands were pulled in various directions. For specific ligands being agonists, the supervised molecular dynamics approach was used to simulate the ligand entry events to the inactive receptor structures. For all investigated receptors the ligand entry/exit pathway goes through the gate between transmembrane helices TM1 and TM7, however, in some cases it combined with a direction toward water milieu.

GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models.

Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand-receptor interactions in visual form.

Aquaporin-graphene interface: relevance to point-of-care device for renal cell carcinoma and desalination.

The aquaporin superfamily of hydrophobic integral membrane proteins constitutes water channels essential to the movement of water across the cell membrane, maintaining homeostatic equilibrium. During the passage of water between the extracellular and intracellular sides of the cell, aquaporins act as ultra-sensitive filters. Owing to their hydrophobic nature, aquaporins self-assemble in phospholipids. If a proper choice of lipids is made then the aquaporin biomimetic membrane can be used in the design of an artificial kidney. In combination with graphene, the aquaporin biomimetic membrane finds practical application in desalination and water recycling using mostly Escherichia coli AqpZ. Recently, human aquaporin 1 has emerged as an important biomarker in renal cell carcinoma. At present, the ultra-sensitive sensing of renal cell carcinoma is cumbersome. Hence, we discuss the use of epitopes from monoclonal antibodies as a probe for a point-of-care device for sensing renal cell carcinoma. This device works by immobilizing the antibody on the surface of a single-layer graphene, that is, as a microfluidic device for sensing renal cell carcinoma.

Approaches for Differentiation and Interconverting GPCR Agonists and Antagonists.

Predicting the functional preferences of the ligands was always a highly demanding task, much harder that predicting whether a ligand can bind to the receptor. This is because of significant similarities of agonists, antagonists and inverse agonists which are binding usually in the same binding site of the receptor and only small structural changes can push receptor toward a particular activation state. For G protein-coupled receptors, due to a large progress in crystallization techniques and also in receptor thermal stabilization, it was possible to obtain a large number of high-quality structures of complexes of these receptors with agonists and non-agonists. Additionally, the long-time-scale molecular dynamics simulations revealed how the activation processes of GPCRs can take place. Using both theoretical and experimental knowledge it was possible to employ many clever and sophisticated methods which can help to differentiate agonists and non-agonists, so one can interconvert them in search of the optimal drug.

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis.

Hydrophobic Ligand Entry and Exit Pathways of the CB1 Cannabinoid Receptor.

It has been reported that some hydrophobic ligands of G-protein-coupled receptors access the receptor's binding site from the membrane rather than from bulk water. In order to identify the most probable ligand entrance pathway into the CB1 receptor, we performed several steered molecular dynamics (SMD) simulations of two CB1 agonists, THC and anandamide, pulling them from the receptor's binding site with constant velocity. The four main directions of ligand pulling were probed: between helices TM4 and TM5, between TM5 and TM6, between TM7 and TM1/TM2, and toward the bulk water. The smallest forces were measured during pulling between TM7 and TM1/TM2. We also performed supervised molecular dynamics (SuMD) simulations for both anandamide and THC entering the CB1 receptor's binding site and found the same pathway as in the pulling simulations. The residues F1742.61 and F1772.64 (both on the TM2 helix) are involved in the gating mechanism and, by forming π-π interactions with ligand molecules, facilitated the ligand orientation required for passage. Using SuMD we also found an alternative binding site for THC. The results of mutagenesis studies evidencing that residues F1742.61 and F1772.64 are important for CB1 ligand binding are in agreement with our observations.

Synthesis of alpha-trifluoromethyl-beta-lactams and esters of beta-amino acids via 1,3-dipolar cycloaddition of nitrones to fluoroalkenes.

Nitrones derived from aromatic or aliphatic aldehydes or ketones react with hexafluoropropene (HFP) or 2H-pentafluoropropene (PFP) to give the respective fluorinated isoxazolidine derivatives in good yields with complete regioselectivity and moderate diastereoselectivity. Catalytic hydrogenolysis of the N-O bond under ambient pressure and temperature leads to fluorides of beta-amino acids that undergo cyclization to alpha-trifluoromethylated beta-lactams or, under acidic conditions, form esters of alpha-trifluoromethylated beta-amino acids.