Molecular Docking of Intrinsically Disordered Proteins: Challenges and Strategies.

Intrinsically disordered proteins (IDPs) are a novel class of proteins that have established a significant importance and attention within a very short period of time. These proteins are essentially characterized by their inherent structural disorder, encoded mainly by their amino acid sequences. The profound abundance of IDPs and intrinsically disordered regions (IDRs) in the biological world delineates their deep-rooted functionality. IDPs and IDRs convey such extensive functionality through their unique dynamic nature, which enables them to carry out huge number of multifaceted biomolecular interactions and make them "interaction hub" of the cellular systems. Additionally, with such widespread functions, their misfunctioning is also intimately associated with multiple diseases. Thus, understanding the dynamic heterogeneity of various IDPs along with their interactions with respective binding partners is an important field with immense potentials in biomolecular research. In this context, molecular docking-based computational approaches have proven to be remarkable in case of ordered proteins. Molecular docking methods essentially model the biomolecular interactions in both structural and energetic terms and use this information to characterize the putative interactions between the two participant molecules. However, direct applications of the conventional docking methods to study IDPs are largely limited by their structural heterogeneity and demands for unique IDP-centric strategies. Thus, in this chapter, we have presented an overview of current methodologies for successful docking operations involving IDPs and IDRs. These specialized methods majorly include the ensemble-based and fragment-based approaches with their own benefits and limitations. More recently, artificial intelligence and machine learning-assisted approaches are also used to significantly reduce the complexity and computational burden associated with various docking applications. Thus, this chapter aims to provide a comprehensive summary of major challenges and recent advancements of molecular docking approaches in the IDP field for their better utilization and greater applicability.Asp (D).

Revealing the key structural features promoting the helical conformation in algal polysaccharide carrageenan in solution.

Carrageenans are industrially important polysaccharides with tunable viscoelastic and gelation properties. The function of polysaccharide depends on its conformation and chemical composition. However, the solution conformations of carrageenans are highly debated, and the structure-function relationship remains elusive. Here, we have studied the intrinsic conformational behavior of a series of carrageenan hexamers in solution, using extensive all-atom classical MD and enhanced sampling. Our findings comprehensively delineate that carrageenans containing the 3,6-anhydrous bridge (κ-C, ι-C, θ-C, and non-sulfated ß-C) adopt compact helical structures as their predominant conformation in solution, whereas carrageenans without the bridge (µ-C, ν-C, and λ-C) remain as extended loosely packed helices; opposing the 'coil-to-helix' paradigm. Glycosidic linkages access a few allowed orientations. We hypothesize that the 3,6-anhydrous bridge, irrespective of carrageenan's sulfation pattern, is essential for stabilizing the helical conformation at the single-chain level. It provides necessary flexibility to the glycosidic linkage to sample conformations close to the experimentally derived helical structure and also prevents the sugar ring flipping. Sulfate groups mainly modify the chain stiffness due to steric and stereo-electronic effects and participate in hydrogen bonding. Such atomistic insights will be helpful for understanding the differential gelation mechanisms of carrageenans and fine-tuning polysaccharide backbone for various industrial applications.

Underpinning beneficial maize response to application of minimally processed homogenates of red and brown seaweeds.

Sap from the fresh seaweed Kappaphycus alvarezii (KA) has been reported to improve crop growth, quality, and stress alleviation. However, limited studies are reported for the minimally processed aqueous homogenates (MPHs) derived from dry seaweeds. The present investigation was envisaged to characterize the MPHs from the red seaweed KA and a brown seaweed Sargassum wightii (SW) and also assess the effect of foliar application on maize (Zea mays) crop performance when applied alone or in proportions ranging from 0% to 100%. Two doses (0.35% and 0.7%) were compared with control. Both the MPHs contained several compounds like retronecine, tyrosyl-glycine, hexyl 2-furoate, 1-phosphatidyl-1D-myo-inositol, 12-(2,3-dihydroxycyclopentyl)-2-dodecanone, and trihomomethionine and many others that have known bioactivity for enhancing plant growth and providing stress tolerance. Both doses of MPHs enhanced crop growth and yield; however, the best response was in general observed at a lower dose. The MPH of SW at 100% gave the highest seed yield at a lower dose, which was also on par with that obtained under a lower dose of 100% KA. Other combinations, 80:20 and 40:60 KA : SW, were also found to give comparable yields. The highest dose of 100% MPH of SW was found on par with control, a phenomenon that was investigated in detail with respect to metabolites and antioxidant profile in leaves as well as membrane modeling. Higher ROS and certain sugar and organic acids were observed in 100% MPH of SW at a higher dose, although none of the antioxidant enzymes were significantly affected, nor was there any change in membrane characteristics of the leaf with respect to control as well as lower dose. Improvements in the seed yield were attributed to improved photosynthate production on account of higher dry matter accumulation in the MPH-treated plants, which may also be attributed to the presence of bioactive compounds in the biostimulants. In the future, it is imperative to direct scientific investigations towards the quantification and identification of the most effective concentrations of these compounds within MPHs to optimize plant responses. The study indicated the beneficial use of the MPHs towards increasing crop production by employing optimum dose as foliar spray to crops.

Phase Transition and Phase Separation in Realistic Thylakoid Lipid Membrane of Marine Algae in All-Atom Simulations.

Thylakoid membranes are specialized membranes predominantly composed of uncommon galacto- and sulfolipids, having distinct roles in photosynthesis. Large acyl chain variety and richness in polyunsaturated fatty acid (PUFA) content of thylakoid lipids further add to the compositional complexity. The function of these membrane systems is intimately dependent on the fluidity of its lipid matrix, which is strongly modulated by the lipid composition and temperature. The present work, employing extensive atomistic simulations, provides the first atomistic view of the phase transition and domain coexistence in a model membrane composed of thylakoid lipids of a commercially important red alga Gracilaria corticata between 10 and 40 °C. The growth and photosynthetic activity of marine algae are greatly influenced by the seawater temperature. So far, little is known about the molecular organization of lipids in thylakoid membranes, in particular their adaptive arrangements under temperature stress. Our simulations show that the algal thylakoid membrane undergoes a transition from a gel-like phase at a low temperature, 10-15 °C, to a homogeneous liquid-crystalline phase at a high temperature, 40 °C. Clear evidence of spontaneous phase separation into coexisting nanoscale domains is detected at intermediate temperatures nearing the optimal growth temperature range. Particularly, at 25-30 °C, we identified the formation of a stable ripple phase, where the gel-like domains rich in saturated and nearly hexagonally packed lipids were separated from fluid-like domains enriched in lipids containing PUFA chains. The phase separation is driven by the spontaneous and preferential segregation of lipids into differentially ordered domains, mainly depending on the acyl chain types. Cholesterol impairs the phase transition and the emergence of domains and induces a fairly uniform liquid-ordered phase in the membrane over the temperatures studied. This work improves the understanding of the properties and reorganization of lipids in the thylakoid membrane in response to temperature variation.

A cholesterol analog stabilizes the human ß2-adrenergic receptor nonlinearly with temperature.

In cell membranes, G protein-coupled receptors (GPCRs) interact with cholesterol, which modulates their assembly, stability, and conformation. Previous studies have shown how cholesterol modulates the structural properties of GPCRs at ambient temperature. Here, we characterized the mechanical, kinetic, and energetic properties of the human ß2-adrenergic receptor (ß2AR) in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) at room temperature (25°C), at physiological temperature (37°C), and at high temperature (42°C). We found that CHS stabilized various structural regions of ß2AR differentially, which changed nonlinearly with temperature. Thereby, the strongest effects were observed for structural regions that are important for receptor signaling. Moreover, at 37°C, but not at 25° or 42°C, CHS caused ß2AR to increase and stabilize conformational substates to adopt to basal activity. These findings indicate that the nonlinear, temperature-dependent action of CHS in modulating the structural and functional properties of this GPCR is optimized for 37°C.

Is Lipid Specificity Key to the Potential Antiviral Activity of Mouthwash Reagent Chlorhexidine against SARS-CoV-2?

Chlorhexidine (CHX), a popular antibacterial drug, is widely used for oral health. Emerging pieces of evidence suggest that commercially available chlorhexidine mouthwash formulations are effective in suppressing the spread of SARS-CoV-2, possibly through destabilization of the viral lipid envelope. CHX is known for its membrane-active properties; however, the molecular mechanism revealing how it damages the viral lipid envelope is yet to be understood. Here we used extensive conventional and umbrella sampling simulations to quantify the effects of CHX on model membranes mimicking the composition of the SARS-CoV-2 outer lipid membrane as well as the host plasma membrane. Our results show that the lipid composition and physical properties of the membrane play an important role in binding and insertion, with CHX binding favorably to the viral membrane over the plasma membrane. Among the simulated lipids, CHX preferentially binds to anionic lipids, PS and PI, which are more concentrated in the viral membrane. The deeper and stable binding of CHX to the viral membrane results in more pronounced swelling of the membrane laterally with a thinning of the bilayer. The overall free energies of pore formation are strongly reduced for the viral membrane compared to the plasma membrane; however, CHX has a larger concentration-dependent effect on free energies of pore formation in the plasma membrane than the viral membrane. The results indicate that CHX is less toxic to the human plasma membrane at low concentrations. Our simulations reveal that CHX facilitates pore formation by the combination of thinning the membrane and accumulation at the water defect. This study provides insights into the mechanism underlying the anti-SARS-CoV-2 potency of CHX, supporting its potential for application as an effective and safe oral rinse agent for preventing viral transmission.

Polyunsaturated Fatty Acid Modulates Membrane-Bound Monomeric α-Synuclein by Modulating Membrane Microenvironment through Preferential Interactions.

There is ample evidence that both native functions and pathogenic aggregation of α-synuclein are intimately dependent on lipid interactions and fatty acid type; the regulatory mechanism however remains unclear. In the present work, using extensive atomistic molecular dynamics simulations and enhanced-sampling, we have focused on exploring the mechanism of fatty acid dependent regulation of monomeric α-Syn100 in a native synaptic vesicle-like membrane. Our results show that α-Syn100 spontaneously binds to the membrane through its N-terminal region (residues 1-34), where the depth of membrane insertion, the structure, and orientation of the membrane-bound α-Syn100 and its impact on membrane structure are modulated by docosahexaenoic acid (DHA). DHA is a polyunsaturated fatty acid abundantly found in the brain and known to promote the oligomerization of α-synuclein. We found that DHA exhibits marked propensity to interact with monomeric α-Syn100 and modulates the microenvironment of the protein by preferentially sorting DHA-containing phospholipids, depleting other phospholipids and cholesterol as well as increasing the proportion of anionic to neutral lipids in the immediate vicinity of the protein. Owing to the unique conformational flexibility, DHA chains form more lipid-packing defects in the membrane and efficiently coat the membrane-embedded surface of the protein, compared to the saturated and monounsaturated fatty acids. DHA thus makes the bilayer more amiable to protein adsorption and less prone to α-synuclein-induced perturbation associated with cytotoxicity. Indeed, in the absence of DHA, we observed significant thinning of the local bilayer membrane induced by α-Syn100. Though α-Syn100 is predominantly α-helical in membranes studied here, in the presence of DHA we observe formation of ß-sheet/ß-strands in the C-terminal region (residues 35-100) of α-Syn100, which is extended out from the membrane surface. Notably, DHA induces ß structure in the NAC domain of α-Syn100 and promotes extended conformations as well as large solvent exposure of this hydrophobic domain, properties that are known to facilitate self-assembly of α-synuclein. To the best of our knowledge, this study for the first time provides the atomistic insights into DHA-induced regulatory mechanism of monomeric α-synuclein, having implications in protein structure and its physiological/pathological functions.

Understanding the Role of Lipids in Signaling Through Atomistic and Multiscale Simulations of Cell Membranes.

Cell signaling controls essentially all cellular processes. While it is often assumed that proteins are the key architects coordinating cell signaling, recent studies have shown more and more clearly that lipids are also involved in signaling processes in a number of ways. Lipids do, for instance, act as messengers, modulate membrane receptor conformation and dynamics, and control membrane receptor partitioning. Further, through structural modifications such as oxidation, the functions of lipids as part of signaling processes can be modified. In this context, in this article we discuss the understanding recently revealed by atomistic and coarse-grained computer simulations of nanoscale processes and underlying physicochemical principles related to lipids' functions in cellular signaling.

Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico.

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.

Long-chain GM1 gangliosides alter transmembrane domain registration through interdigitation.

Extracellular and cytosolic leaflets in cellular membranes are distinctly different in lipid composition, yet they contribute together to signaling across the membranes. Here we consider a mechanism based on long-chain gangliosides for coupling the extracellular and cytosolic membrane leaflets together. Based on atomistic molecular dynamics simulations, we find that long-chain GM1 in the extracellular leaflet exhibits a strong tendency to protrude into the opposing bilayer leaflet. This interdigitation modulates the order in the cytosolic monolayer and thereby strengthens the interaction and coupling across a membrane. Coarse-grained simulations probing longer time scales in large membrane systems indicate that GM1 in the extracellular leaflet modulates the phase behavior in the cytosolic monolayer. While short-chain GM1 maintains phase-symmetric bilayers with a strong membrane registration effect, the situation is altered with long-chain GM1. Here, the significant interdigitation induced by long-chain GM1 modulates the behavior in the cytosolic GM1-free leaflet, weakening and slowing down the membrane registration process. The observed physical interaction mechanism provides a possible means to mediate or foster transmembrane communication associated with signal transduction.

Membrane cholesterol access into a G-protein-coupled receptor.

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

Mechanism of allosteric regulation of ß2-adrenergic receptor by cholesterol.

There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human ß2-adrenergic receptor (ß2AR) - a prototypical G protein-coupled receptor - is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates ß2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5-7 of the receptor. The alternative mechanism, where the ß2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of ß2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.

Indocyanine Green-Loaded Liposomes for Light-Triggered Drug Release.

Light-triggered drug delivery systems enable site-specific and time-controlled drug release. In previous work, we have achieved this with liposomes containing gold nanoparticles in the aqueous core. Gold nanoparticles absorb near-infrared light and release the energy as heat that increases the permeability of the liposomal bilayer, thus releasing the contents of the liposome. In this work, we replaced the gold nanoparticles with the clinically approved imaging agent indocyanine green (ICG). The ICG liposomes were stable at storage conditions (4-22 °C) and at body temperature, and fast near-infrared (IR) light-triggered drug release was achieved with optimized phospholipid composition and a 1:50 ICG-to-lipid molar ratio. Encapsulated small molecular calcein and FITC-dextran (up to 20 kDa) were completely released from the liposomes after light exposure for 15 s. Location of ICG in the PEG layer of the liposomes was simulated with molecular dynamics. ICG has important benefits as a light-triggering agent in liposomes: fast content release, improved stability, improved possibility of liposomal size control, regulatory approval to use in humans, and the possibility of imaging the in vivo location of the liposomes based on the fluorescence of ICG. Near-infrared light used as a triggering mechanism has good tissue penetration and safety. Thus, ICG liposomes are an attractive option for light-controlled and efficient delivery of small and large drug molecules.

How To Minimize Artifacts in Atomistic Simulations of Membrane Proteins, Whose Crystal Structure Is Heavily Engineered: ß2-Adrenergic Receptor in the Spotlight.

Atomistic molecular dynamics (MD) simulations are used extensively to elucidate membrane protein properties. These simulations are based on three-dimensional protein structures that in turn are often based on crystallography. The protein structures resolved in crystallographic studies typically do not correspond to pristine proteins, however. Instead the crystallized proteins are commonly engineered, including structural modifications (mutations, replacement of protein sequences by antibodies, bound ligands, etc.) whose impact on protein structure and dynamics is largely unknown. Here we explore this issue through atomistic MD simulations (∼5 µs in total), focusing on the ß2-adrenergic receptor (ß2AR) that is one of the most studied members of the G-protein coupled receptor superfamily. Starting from an inactive-state crystal structure of ß2AR, we remove the many modifications in ß2AR systematically one at a time, in six consecutive steps. After each step, we equilibrate the system and simulate it quite extensively. The results of this step-by-step approach highlight that the structural modifications used in crystallization can affect ligand and G-protein binding sites, packing at the transmembrane-helix interface region, and the dynamics of connecting loops in ß2AR. When the results of the systematic step-by-step approach are compared to an all-at-once technique where all modifications done on ß2AR are removed instantaneously at the same time, it turns out that the step-by-step method provides results that are superior in terms of maintaining protein structural stability. The results provide compelling evidence that for membrane proteins whose 3D structure is based on structural engineering, the preparation of protein structure for atomistic MD simulations is a delicate and sensitive process. The results show that most valid results are found when the structural modifications are reverted slowly, one at a time.

Insights into the behavioral difference of water in the presence of GM1.

Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface.

Cholesterol under oxidative stress-How lipid membranes sense oxidation as cholesterol is being replaced by oxysterols.

The behavior of oxysterols in phospholipid membranes and their effects on membrane properties were investigated by means of dynamic light scattering, fluorescence spectroscopy, NMR, and extensive atomistic simulations. Two families of oxysterols were scrutinized-tail-oxidized sterols, which are mostly produced by enzymatic processes, and ring-oxidized sterols, formed mostly via reactions with free radicals. The former family of sterols was found to behave similar to cholesterol in terms of molecular orientation, roughly parallel to the bilayer normal, leading to increasing membrane stiffness and suppression of its membrane permeability. In contrast, ring-oxidized sterols behave quantitatively differently from cholesterol. They acquire tilted orientations and therefore disrupt the bilayer structure with potential implications for signaling and other biochemical processes in the membranes.

Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate.

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.

Enzymatic oxidation of cholesterol: properties and functional effects of cholestenone in cell membranes.

Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone) has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone.

How well does cholesteryl hemisuccinate mimic cholesterol in saturated phospholipid bilayers?

Cholesteryl hemisuccinate is a detergent that is often used to replace cholesterol in crystallization of membrane proteins. Here we employ atomistic molecular dynamics simulations to characterize how well the properties of cholesteryl hemisuccinate actually match those of cholesterol in saturated protein-free lipid membranes. We show that the protonated form of cholesteryl hemisuccinate mimics many of the membrane properties of cholesterol quite well, while the deprotonated form of cholesteryl hemisuccinate is less convincing in this respect. Based on the results, we suggest that cholesteryl hemisuccinate in its protonated form is a quite faithful mimic of cholesterol for membrane protein crystallization, if specific cholesterol-protein interactions (not investigated here) are not playing a crucial role.

The challenges of understanding glycolipid functions: An open outlook based on molecular simulations.

Glycolipids are the most complex lipid type in cell membranes, characterized by a great diversity of different structures and functions. The underlying atomistic/molecular interactions and mechanisms associated with these functions are not well understood. Here we discuss how atomistic and molecular simulations can be used to shed light on the role of glycolipids in membrane structure and dynamics, receptor function, and other phenomena related to emergence of diseases such as Parkinson's. The cases we discuss highlight the challenge to understand how glycolipids function in cell membranes, and the significant added value that one would gain by bridging molecular simulations with experiments. This article is part of a Special Issue entitled Tools to study lipid functions.