Fentanyl and the Fluorinated Fentanyl Derivative NFEPP Elicit Distinct Hydrogen-Bond Dynamics of the Opioid Receptor.

The development of safe therapeutics to manage pain is of central interest for biomedical applications. The fluorinated fentanyl derivative N-(3-fluoro-1-phenethylpiperidin-4-yl)-N-phenylpropionamide (NFEPP) is potentially a safer alternative to fentanyl because unlike fentanyl─which binds to the µ-opioid receptor (MOR) at both physiological and acidic pH─NFEPP might bind to the MOR only at acidic pH typical of inflamed tissue. Knowledge of the protonation-coupled dynamics of the receptor-drug interactions is thus required to understand the molecular mechanism by which receptor activation initiates cell signaling to silence pain. To this end, here we have carried out extensive atomistic simulations of the MOR in different protonation states, in the absence of opioid drugs, and in the presence of fentanyl vs NFEPP. We used graph-based analyses to characterize internal hydrogen-bond networks that could contribute to the activation of the MOR. We find that fentanyl and NFEPP prefer distinct binding poses and that, in their binding poses, fentanyl and NFEPP partake in distinct internal hydrogen-bond networks, leading to the cytoplasmic G-protein-binding region. Moreover, the protonation state of functionally important aspartic and histidine side chains impacts hydrogen-bond networks that extend throughout the receptor, such that the ligand-bound MOR presents at its cytoplasmic G-protein-binding side, a hydrogen-bonding environment where dynamics depend on whether fentanyl or NFEPP is bound, and on the protonation state of specific MOR groups. The exquisite sensitivity of the internal protein-water hydrogen-bond network to the protonation state and to details of the drug binding could enable the MOR to elicit distinct pH- and opioid-dependent responses at its cytoplasmic G-protein-binding site.

pH-triggered pore-forming peptides with strong composition-dependent membrane selectivity.

Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pH-dependent delivery peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration but only at pH < ∼6. Further advancements will also require better selectivity for specific membranes. Here, we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pH-dependent delivery peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from phosphatidylcholine with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness and does not occur at any peptide concentration in fluid bilayers made from phosphatidylcholine lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane-spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.

Interactive Interface for Graph-Based Analyses of Dynamic H-Bond Networks: Application to Spike Protein S.

Dynamic hydrogen-bond networks are key determinants of protein conformational dynamics. In the case of macromolecular protein complexes, which can have a large number of hydrogen bonds giving rise to extensive hydrogen-bond networks, efficient algorithms are required to analyze interactions that could be important for the dynamics and biological function of the complex. We present here a highly efficient, standalone interface designed for analyses of dynamical hydrogen-bond networks of biomolecules and macromolecular complexes. To facilitate a comprehensive description of protein dynamics, the interface includes analyses of hydrophobic interactions. We illustrate the usefulness and workflow of the interface by dissecting the dynamics of the ectodomain of SARS-CoV-2 protein S in its closed conformation. We find that protein S contains numerous local clusters of dynamic hydrogen bonds and identify hydrogen bonds that are sampled persistently. The receptor binding domain of the spike protein hosts only a handful of persistent hydrogen-bond clusters, suggesting structural plasticity. Our data analysis interface is released here for open use.

C-Graphs Tool with Graphical User Interface to Dissect Conserved Hydrogen-Bond Networks: Applications to Visual Rhodopsins.

Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central interest to understand protein reaction mechanisms. Here, we present C-Graphs, an efficient tool with graphical user interface that analyzes data sets of static protein structures or of independent numerical simulations to identify conserved, vs unique, hydrogen bonds and hydrogen-bond networks. For static structures, which may belong to the same protein or to proteins with different sequences, C-Graphs uses a clustering algorithm to identify sites of the hydrogen-bond network where waters are conserved among the structures. Using C-Graphs, we identify an internal protein-water hydrogen-bond network common to static structures of visual rhodopsins and adenosine A2A G protein-coupled receptors (GPCRs). Molecular dynamics simulations of a visual rhodopsin indicate that the conserved hydrogen-bond network from static structure can recruit dynamic hydrogen bonds and extend throughout most of the receptor. We release with this work the code for C-Graphs and its graphical user interface.

CHARMM Force-Field Parameters for Morphine, Heroin, and Oliceridine, and Conformational Dynamics of Opioid Drugs.

Opioid drug binding to specialized G protein-coupled receptors (GPCRs) can lead to analgesia upon activation via downstream Gi protein signaling and to severe side effects via activation of the ß-arrestin signaling pathway. Knowledge of how different opioid drugs interact with receptors is essential, as it can inform and guide the design of safer therapeutics. We performed quantum and classical mechanical computations to explore the potential energy landscape of four opioid drugs: morphine and its derivatives heroin and fentanyl and for the unrelated oliceridine. From potential energy profiles for bond twists and from interactions between opioids and water, we derived a set of force-field parameters that allow a good description of structural properties and intermolecular interactions of the opioids. Potential of mean force profiles computed from molecular dynamics simulations indicate that fentanyl and oliceridine have complex energy landscapes with relatively small energy penalties, suggesting that interactions with the receptor could select different binding poses of the drugs.

Reactions at Biomembrane Interfaces.

Membranes surrounding the biological cell and its internal compartments host proteins that catalyze chemical reactions essential for the functioning of the cell. Rather than being a passive structural matrix that holds membrane-embedded proteins in place, the membrane can largely shape the conformational energy landscape of membrane proteins and impact the energetics of their chemical reaction. Here, we highlight the challenges in understanding how lipids impact the conformational energy landscape of macromolecular membrane complexes whose functioning involves chemical reactions including proton transfer. We review here advances in our understanding of how chemical reactions occur at membrane interfaces gleaned with both theoretical and experimental advances using simple protein systems as guides. Our perspective is that of bridging experiments with theory to understand general physicochemical principles of membrane reactions, with a long term goal of furthering our understanding of the role of the lipids on the functioning of complex macromolecular assemblies at the membrane interface.

Opioid Receptors and Protonation-Coupled Binding of Opioid Drugs.

Opioid receptors are G-protein-coupled receptors (GPCRs) part of cell signaling paths of direct interest to treat pain. Pain may associate with inflamed tissue characterized by acidic pH. The potentially low pH at tissue targeted by opioid drugs in pain management could impact drug binding to the opioid receptor, because opioid drugs typically have a protonated amino group that contributes to receptor binding, and the functioning of GPCRs may involve protonation change. In this review, we discuss the relationship between structure, function, and dynamics of opioid receptors from the perspective of the usefulness of computational studies to evaluate protonation-coupled opioid-receptor interactions.

Protein-water hydrogen-bond networks of G protein-coupled receptors: Graph-based analyses of static structures and molecular dynamics.

Protein and protein-water hydrogen bonds shape the conformational energy landscape of G Protein-Coupled Receptors, GPCRs. As numerous static structures of GPCRs have been solved, the important question arises whether GPCR structures and GPCR conformational dynamics could be described in terms of conserved hydrogen-bond networks, and alterations of these hydrogen-bond networks along the reaction coordinate of the GPCR. To enable efficient analyses of the hydrogen-bond networks of GPCRs we implemented graph-based algorithms, and applied these algorithms to static GPCR structures from structural biology, and from molecular dynamics simulations of two opioid receptors. We find that static GPCR structures tend to have a conserved, core hydrogen-bond network which, when protein and water dynamics are included with simulations, extends to comprise most of the interior of an inactive receptor. In an active receptor, the dynamic protein-water hydrogen-bond network spans the entire receptor, bridging all functional motifs. Such an extensive, dynamic hydrogen-bond network might contribute to the activation mechanism of the GPCR.

A graph-based approach identifies dynamic H-bond communication networks in spike protein S of SARS-CoV-2.

Corona virus spike protein S is a large homo-trimeric protein anchored in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, and contribute symmetrically to a relatively large H-bond cluster at the receptor binding domain, and to a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.

Potential energy function for a photo-switchable lipid molecule.

Photo-switchable lipids are synthetic lipid molecules used in photo-pharmacology to alter membrane lateral pressure and thus control opening and closing of mechanosensitive ion channels. The molecular picture of how photo-switchable lipids interact with membranes or ion channels is poorly understood. To facilitate all-atom simulations that could provide a molecular picture of membranes with photo-switchable lipids, we derived force field parameters for atomistic computations of the azobenzene-based fatty acid FAAzo-4. We implemented a Phyton-based algorithm to make the optimization of atomic partial charges more efficient. Overall, the parameters we derived give good description of the equilibrium structure, torsional properties, and non-bonded interactions for the photo-switchable lipid in its trans and cis intermediate states, and crystal lattice parameters for trans-FAAzo-4. These parameters can be extended to all-atom descriptions of various photo-switchable lipids that have an azobenzene moiety.

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes.

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4 CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pKa values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.

Phosphatidylglyerol Lipid Binding at the Active Site of an Intramembrane Protease.

Transmembrane substrate cleavage by the small Escherichia coli rhomboid protease GlpG informs on mechanisms by which lipid interactions shape reaction coordinates of membrane-embedded enzymes. Here, I review and discuss new work on the molecular picture of protein-lipid interactions that might govern the formation of the substrate-enzyme complex in fluid lipid membranes. Negatively charged PG-type lipids are of particular interest, because they are a major component of bacterial membranes. Atomistic computer simulations indicate POPG and DOPG lipids bridge remote parts of GlpG and might pre-occupy the substrate-docking site. Inhibition of catalytic activity by PG lipids could arise from ligand-like lipid binding at the active site, which could delay or prevent substrate docking. Dynamic protein-lipid H-bond networks, water access to the active site, and fluctuations in the orientation of GlpG suggest that GlpG has lipid-coupled dynamics that could shape the energy landscape of transmembrane substrate docking.

Potential Energy Function for Fentanyl-Based Opioid Pain Killers.

Opioids are molecules whose binding to specialized G-Protein Coupled Receptors (GPCRs) triggers a signaling cascade that leads to the downregulation of pain pathways. Binding of an opioid to the membrane-embedded GPCR occurs when the opioid molecule is protonated, which provides a potential strategy to design nontoxic opioids that are protonated and bind to the GPCR only at the low pH of injured or inflamed tissue. Excellent model systems to study protonation-dependent binding of opioids to GPCRs are fentanyl, which is protonated and binds to the GPCR at both physiological and low pH, and the fluorinated fentanyl derivative NFEPP, which is protonated and binds to the GPCR only at low pH. The molecular mechanisms of fentanyl and NFEPP binding to the GPCR are largely unknown. To enable atomistic studies of opioid binding to GPCRs, we have carried out extensive quantum mechanical and classical mechanical computations to derive a potential energy function for fentanyl and NFEPP and present force field parameters for both opioid molecules. We find that fluorination alters the electronic ground state properties of fentanyl. As a consequence, fentanyl and NFEPP have distinct torsional and electrostatic properties likely to impact how they bind to receptors.

Membrane cholesterol as regulator of human rhomboid protease RHBDL4.

In the last decade, intramembrane proteases have gained increasing attention because of their many links to various diseases. Nevertheless, our understanding as to how they function or how they are regulated is still limited, especially when it comes to human homologues. In this regard, here we sought to unravel mechanisms of regulation of the protease rhomboid-like protein-4 (RHBDL4), one of five active human serine intramembrane proteases. In view of our recent finding that human RHBDL4 efficiently cleaves the amyloid precursor protein (APP), a key protein in the pathology of Alzheimer's disease, we used established reagents to modulate the cellular cholesterol content and analyzed the effects of this modulation on RHBDL4-mediated processing of endogenous APP. We discovered that lowering membrane cholesterol levels increased the levels of RHBDL4-specific endogenous APP fragments, whereas high cholesterol levels had the opposite effect. Direct binding of cholesterol to APP did not mediate these modulating effects of cholesterol. Instead, using homology modeling, we identified two potential cholesterol-binding motifs in the transmembrane helices 3 and 6 of RHBDL4. Substitution of the essential tyrosine residues of the potential cholesterol-binding motifs to alanine increased the levels of endogenous APP C-terminal fragments, reflecting enhanced RHBDL4 activity. In summary, we provide evidence that the activity of RHBDL4 is regulated by cholesterol likely through a direct binding of cholesterol to the enzyme.

Using Graphs of Dynamic Hydrogen-Bond Networks To Dissect Conformational Coupling in a Protein Motor.

DExD/H-box proteins are soluble enzymes that couple binding and hydrolysis of adenosine triphosphate (ATP) with reactions involving RNA metabolism or bind and push newly synthesized proteins across bacterial cell membranes. Knowledge of the reaction mechanism of these enzymes could help the development of new therapeutics. In order to explore the mechanism of long-distance conformational coupling in SecA, the DEAD-box motor of the Sec protein secretion in bacteria, we implemented algorithms that provide simplified graph representations of the protein's dynamic hydrogen-bond networks. We find that mutations near the nucleotide-binding site or changes of the nucleotide-binding state of SecA associate with altered dynamics at the preprotein binding domain and identify extended networks of hydrogen bonds that connect the active site of SecA to the region where SecA binds newly synthesized secretory proteins. Water molecules participate in hydrogen-bonded water chains that bridge functional domains of SecA and could contribute to long-distance conformational coupling.

Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II.

Proton-transfer proteins are often exposed to the bulk clusters of carboxylate groups that might bind protons transiently. This raises important questions as to how the carboxylate groups of a protonated cluster interact with each other and with water, and how charged protein groups and hydrogen-bonded waters could have an impact on proton transfers at the cluster. We address these questions by combining classical mechanical and quantum mechanical computations with the analysis of cyanobacterial photosystem II crystal structures from Thermosynechococcus elongatus. The model system we use consists of an interface between PsbO and PsbU, which are two extrinsic proteins of photosystem II. We find that a protonated carboxylate pair of PsbO is part of a dynamic network of protein-water hydrogen bonds which extends across the protein interface. Hydrogen-bonded waters and a conserved lysine sidechain largely shape the energetics of proton transfer at the carboxylate cluster.

Biophysical mechanism of rhomboid proteolysis: Setting a foundation for therapeutics.

Rhomboid proteolysis is an important cellular process that involves cleavage of transmembrane substrates by membrane-embedded rhomboid proteases. Such proteases can cleave substrates with essential roles in the cell, and their action has been implicated in human diseases. The remarkable sensitivity of rhomboid proteolysis to the lipid membrane environment highlights the usefulness of rhomboid proteases as model systems to dissect the role of lipids in reaction mechanisms of membrane proteins, including cleavage of substrates by γ-secretase. I discuss here the hydrogen-bond networks likely important for the reaction mechanism of rhomboid proteases, and how the composition of the lipid membrane can alter protein dynamics.

Motions of the SecA protein motor bound to signal peptide: Insights from molecular dynamics simulations.

SecA is an essential part of the Sec pathway for protein secretion in bacteria. In this pathway, SecA interacts with the N-terminal fragment of the secretory protein - the signal peptide, and couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the SecY protein translocon. How interactions with the signal peptide alter the conformational dynamics and long-distance conformational couplings of SecA is a key open question that we address here with molecular dynamics techniques. Analyses of protein motions indicate that the signal peptide alters SecA dynamics not only at the site where this peptide binds, but also at a nucleotide-binding domain. Hydrogen bond clusters contribute to the long-distance propagation of changes in SecA dynamics.

Revised CHARMM force field parameters for iron-containing cofactors of photosystem II.

Photosystem II is a complex protein-cofactor machinery that splits water molecules into molecular oxygen, protons, and electrons. All-atom molecular dynamics simulations have the potential to contribute to our general understanding of how photosystem II works. To perform reliable all-atom simulations, we need accurate force field parameters for the cofactor molecules. We present here CHARMM bonded and non-bonded parameters for the iron-containing cofactors of photosystem II that include a six-coordinated heme moiety coordinated by two histidine groups, and a non-heme iron complex coordinated by bicarbonate and four histidines. The force field parameters presented here give water interaction energies and geometries in good agreement with the quantum mechanical target data. © 2017 Wiley Periodicals, Inc.

Dynamic Water Hydrogen-Bond Networks at the Interface of a Lipid Membrane Containing Palmitoyl-Oleoyl Phosphatidylglycerol.

Lipid membrane interfaces are complex environments that host essential cellular processes such as binding of proteins or drug molecules. A key open question is how water molecules at the interface of membranes with anionic lipids participate in protein binding. To address this question, we studied the dynamics of water hydrogen bonding at the interface of membranes composed of phosphatidylcholine and phosphatidylglycerol, and implemented an algorithm to identify hydrogen-bonded networks at the interface of a lipid membrane, and to characterize their dynamics and linear connections. We find that the membrane interface is characterized by a rich network of hydrogen-bonded water chains that bridge lipid headgroups, some of which form transient lipid clusters. Water-mediated bridges between with lipid phosphate groups are dynamic, with residence lifetimes on the order of picoseconds. These clusters of water/lipid headgroup hydrogen bonds could provide a platform for the binding of proteins or of drug molecules with cationic groups.