Lipid redistribution in the highly curved footprint of Piezo1.

We investigate the effects on the distribution of lipids in the plasma membrane that are caused by the insertion of a protein, Piezo1, that significantly distorts the membrane toward the cytosol. From coarse-grained molecular dynamics simulations, we find that the major effects occur in the outer, extracellular, leaflet. The mol fraction of cholesterol increases significantly in the curved region of the membrane close to Piezo1, while those of phosphatidylcholine and of sphingomyelin decrease. In the inner leaflet, mol fractions of cholesterol and of phosphatidylethanolamine decrease slightly as the protein is approached, while that of phosphatidylserine increases slightly. The mol fraction of phosphatidylcholine decreases markedly as the protein is approached. Most of these results are understood in the context of a theoretical model that utilizes two elements: 1) a coupling between the leaflets' actual curvatures and their compositionally dependent spontaneous curvatures and 2) the dependence of the spontaneous curvatures not only on the mol fractions of the phospholipids, but also on the effect that cholesterol has on the spontaneous curvatures of the phospholipids.

Characterizing the lipid fingerprint of the mechanosensitive channel Piezo2.

Piezo2 is a mechanosensitive ion channel that plays critical roles in sensing touch and pain, proprioception, and regulation of heart rate. Global knockout of Piezo2 leads to perinatal lethality in mice, and Piezo2 gain-of-function mutations are associated with distal arthrogryposis, a disease characterized by congenital joint contractures. Emerging evidence suggests that Piezo channels (Piezo1 and Piezo2) can be regulated by their local membrane environment and particularly by cholesterol and phosphoinositides. To characterize the local Piezo2 lipid environment and investigate key lipid-protein interactions, we carried out coarse-grained molecular dynamics simulations of Piezo2 embedded in a complex mammalian membrane containing >60 distinct lipid species. We show that Piezo2 alters its local membrane composition such that it becomes enriched with specific lipids, such as phosphoinositides, and forms specific, long-term interactions with a variety of lipids at functionally relevant sites.

Computational studies of Piezo1 yield insights into key lipid-protein interactions, channel activation, and agonist binding.

Piezo1 is a mechanically gated ion channel responsible for converting mechanical stimuli into electrical signals in mammals, playing critical roles in vascular development and blood pressure regulation. Dysfunction of Piezo1 has been linked to several disorders, including hereditary xerocytosis (gain-of-function) and generalised lymphatic dysplasia (loss-of-function), as well as a common polymorphism associated with protection against severe malaria. Despite the important physiological roles played by Piezo1, its recent discovery means that many aspects underlying its function are areas of active research. The recently elucidated cryo-EM structures of Piezo1 have paved the way for computational studies, specifically molecular dynamic simulations, to examine the protein's behaviour at an atomistic level. These studies provide valuable insights to Piezo1's interactions with surrounding membrane lipids, a small-molecule agonist named Yoda1, and Piezo1's activation mechanisms. In this review, we summarise and discuss recent papers which use computational techniques in combination with experimental approaches such as electrophysiology/mutagenesis studies to investigate Piezo1. We also discuss how to mitigate some shortcomings associated with using computational techniques to study Piezo1 and outline potential avenues of future research.

Initiating Coarse-Grained MD Simulations for Membrane-Bound Proteins.

Molecular dynamics (MD) simulations have become a widely used tool in the scientific community for understanding molecular scale phenomena that are challenging to address with wet-lab techniques. Coarse-grained simulations, in which multiple atoms are combined into single beads, allow for larger systems and longer time scales to be explored than atomistic techniques. Here, we describe the procedures and equipment required to set up coarse-grained simulations of membrane-bound proteins in a lipid bilayer that can mimic many membrane environments.

Differences in local anaesthetic and antiepileptic binding in the inactivated state of human sodium channel Nav1.4.

Voltage-gated sodium channels play a vital role in nerve and muscle cells, enabling them to encode and transmit electrical signals. Currently, there exist several classes of drugs that aim to inhibit these channels for therapeutic purposes, including local anesthetics, antiepileptics and antiarrhythmics. However, sodium-channel-inhibiting drugs lack subtype specificity; instead, they inhibit all sodium channels in the human body. Improving understanding of the mechanisms of binding of existing nonselective drugs is important in providing insight into how subtype-selective drugs could be developed. This study used molecular dynamics simulations to investigate the binding of the antiepileptics carbamazepine and lamotrigine and the local anesthetic lidocaine in neutral and charged states to the recently resolved human Nav1.4 channel. Replica exchange solute tempering was used to enable greater sampling of each compound within the pore. It was found that all four compounds show similarities in their binding sites within the pore. However, the positions of the carbamazepine and lamotrigine did not occlude the center of the pore but preferentially bound to homologous domain DII and DIII. The charged and neutral forms of lidocaine positioned themselves more centrally in the pore, with more common interactions with DIV. The best localized binding site was for charged lidocaine, whose aromatic moiety interacted with Y1593, whereas the amine projected toward the selectivity filter. Comparisons with our previous simulations and published structures highlight potential differences between tonic and use-dependent block related to conformational changes occurring in the pore.

In pursuit of a selective hepatocellular carcinoma therapeutic agent: Novel thalidomide derivatives with antiproliferative, antimigratory and STAT3 inhibitory properties.

Advanced stage liver cancer is predominantly treated with the multi-kinase inhibitor sorafenib; however, this therapeutic agent lacks selectivity in its cytotoxic actions and is associated with poor survival outcomes. Herein we report the design and preparation of several thalidomide derivatives, including a variety of novel thioether-containing forms that are especially rare in the literature. Importantly, two of the derivatives described are potent antiproliferative agents with dose-dependent selectivity for tumorigenic liver progenitor cells (LPC) growth inhibition (up to 36% increase in doubling time at 10 µM) over non-tumorigenic cells (no effect at 10 µM). Furthermore, these putative anti-liver cancer agents were also found to be potent inhibitors of tumorigenic LPC migration. This report also describes these derivatives' effects on several key signalling pathways in our novel liver cell lines by immunofluorescence and AlphaLISA assays. Aryl thioether derivative 7f significantly reduced STAT3 phosphorylation (23%) and its nuclear localisation (16%) at 10 µM in tumorigenic LPCs, implicating the IL-6/JAK/STAT3 axis is central in the mode of action of our derivatives.

Piezo1 Forms Specific, Functionally Important Interactions with Phosphoinositides and Cholesterol.

Touch, hearing, and blood pressure regulation require mechanically gated ion channels that convert mechanical stimuli into electrical currents. One such channel is Piezo1, which plays a key role in the transduction of mechanical stimuli in humans and is implicated in diseases, such as xerocytosis and lymphatic dysplasia. There is building evidence that suggests Piezo1 can be regulated by the membrane environment, with the activity of the channel determined by the local concentration of lipids, such as cholesterol and phosphoinositides. To better understand the interaction of Piezo1 with its environment, we conduct simulations of the protein in a complex mammalian bilayer containing more than 60 different lipid types together with electrophysiology and mutagenesis experiments. We find that the protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. We also identify a number of key structural connections between the propeller and pore domains located close to lipid-binding sites.

Protonation state of inhibitors determines interaction sites within voltage-gated sodium channels.

Voltage-gated sodium channels are essential for carrying electrical signals throughout the body, and mutations in these proteins are responsible for a variety of disorders, including epilepsy and pain syndromes. As such, they are the target of a number of drugs used for reducing pain or combatting arrhythmias and seizures. However, these drugs affect all sodium channel subtypes found in the body. Designing compounds to target select sodium channel subtypes will provide a new therapeutic pathway and would maximize treatment efficacy while minimizing side effects. Here, we examine the binding preferences of nine compounds known to be sodium channel pore blockers in molecular dynamics simulations. We use the approach of replica exchange solute tempering (REST) to gain a more complete understanding of the inhibitors' behavior inside the pore of NavMs, a bacterial sodium channel, and NavPas, a eukaryotic sodium channel. Using these simulations, we are able to show that both charged and neutral compounds partition into the bilayer, but neutral forms more readily cross it. We show that there are two possible binding sites for the compounds: (i) a site on helix 6, which has been previously determined by many experimental and computational studies, and (ii) an additional site, occupied by protonated compounds in which the positively charged part of the drug is attracted into the selectivity filter. Distinguishing distinct binding poses for neutral and charged compounds is essential for understanding the nature of pore block and will aid the design of subtype-selective sodium channel inhibitors.

Voltage-Gated Sodium Channel Pharmacology: Insights From Molecular Dynamics Simulations.

Voltage-gated ion channels are the target of a range of naturally occurring toxins and therapeutic drugs. There is a great interest in better understanding how these diverse compounds alter channel function in order to design the next generation of therapeutics that can selectively target one of the channel subtypes found in the body. Since the publication of a number of bacterial sodium channel structures, molecular dynamics simulations have been invaluable in gaining a high resolution understanding where many of these small molecules and toxins bind to the channels, how they find their binding site, and how they can selectively bind to one channel subtype over another. This chapter summarizes these recent studies to highlight what has been learnt about channel pharmacology using computer simulations and to draw out shared conclusions, focusing separately on toxin-channel interactions and small molecule-channel interactions.

Multiscale Simulations Suggest a Mechanism for the Association of the Dok7 PH Domain with PIP-Containing Membranes.

Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the ß1/ß2, ß3/ß4, and ß5/ß6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.

Primary and secondary dimer interfaces of the fibroblast growth factor receptor 3 transmembrane domain: characterization via multiscale molecular dynamics simulations.

Receptor tyrosine kinases are single-pass membrane proteins that form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. Fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of the cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface that is more highly populated in heterodimer and mutant configurations that may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer to allow interactions of the arginine side chain with lipid headgroup phosphates.

Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices.

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analyzed via coarse grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations, we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of a family of helix sequences. We illustrate this software via analyses of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analyses of these ensembles of simulations, we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application, we use CGMD simulations to examine the self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase and analyze the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers a proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins.

ReplicOpter: a replicate optimizer for flexible docking.

We present a computationally efficient method for flexible refinement of docking predictions that reflects observed motions within a protein's structural class. Using structural homologs, we derive deformation models that capture likely motions. The models or "replicates" typically align along a rigid core, with a handful of flexible loops, linkers and tails. A few replicates can generate a much larger number of conformers, by exchanging each flexible region independently of the others. In this way, 10 replicates of a protein having 6 flexible regions can be used to generate a million conformations of a molecule. While this has obvious advantages in terms of sampling, the cost of assessing energies at every conformer is prohibitive, particularly when both molecules are flexible. Our approach addresses this combinatorial explosion, using key assumptions to compress the sampling by many orders of magnitude. ReplicOpter can perform hierarchical clustering from a list of rigid docking predictions and find nearby structures to any promising cluster representatives. These predicted complexes can then be refined and rescored. ReplicOpter's scoring function includes a Lennard-Jones potential softened using the Anderson-Chandler-Weeks decomposition, a desolvation term derived from the Atomic Contact Energy function, Coulombic electrostatics, hydrogen bonding, and terms to model pi-pi and pi-cation interactions. ReplicOpter has performed well on several recent CAPRI systems. We are presently benchmarking ReplicOpter on the complete docking benchmark set to fully establish its utility in refining rigid docking predictions and identifying near-native solutions.