Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions.

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

Mechanistic origin of partial agonism of tetrahydrocannabinol for cannabinoid receptors.

Cannabinoid receptor 1 (CB1) is a therapeutically relevant drug target for controlling pain, obesity, and other central nervous system disorders. However, full agonists and antagonists of CB1 have been reported to cause serious side effects in patients. Therefore, partial agonists have emerged as a viable alternative as they can mitigate overstimulation and side effects. One of the key bottlenecks in the design of partial agonists, however, is the lack of understanding of the molecular mechanism of partial agonism itself. In this study, we examine two mechanistic hypotheses for the origin of partial agonism in cannabinoid receptors and predict the mechanistic basis of partial agonism exhibited by Δ9-Tetrahydrocannabinol (THC) against CB1. In particular, we inspect whether partial agonism emerges from the ability of THC to bind in both agonist and antagonist-binding poses or from its ability to only partially activate the receptor. We used extensive molecular dynamics simulations and Markov state modeling to capture the THC binding in both antagonist and agonist-binding poses in the CB1 receptor. Furthermore, we predict that binding of THC in the agonist-binding pose leads to rotation of toggle switch residues and causes partial outward movement of intracellular transmembrane helix 6 (TM6). Our simulations also suggest that the alkyl side chain of THC plays a crucial role in determining partial agonism by stabilizing the ligand in the agonist and antagonist-like poses within the pocket. Taken together, this study provides important insights into the mechanistic origin of the partial agonism of THC.

Identification of sulfonamide compounds active on the insect nervous system: Molecular modeling, synthesis and biological evaluation.

Insect nicotinic acetylcholine receptors (nAChRs) are a recognized target for insecticide design. In this work, we have identified, from a structure-based approach using molecular modeling tools, ligands with potential selective activity for pests versus pollinators. A high-throughput virtual screening with the Openeye software was performed using a library from the ZINC database, thiacloprid being used as the target structure. The top sixteen molecules were then docked in α6 cockroach and honeybee homomeric nAChRs to check from a theoretical point of view relevant descriptors in favor of pest selectivity. Among the selected molecules, one original sulfonamide compound has afterward been synthesized, together with various analogs. Two compounds of this family have been shown to behave as activators of the cockroach cholinergic synaptic transmission.

The substrate import mechanism of the human serotonin transporter.

The serotonin transporter (SERT) initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. The cryogenic electron microscopy structures of SERT bound to ibogaine and the physiological substrate serotonin resolved in different states have provided a glimpse of the functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to intermediate states are not fully understood. Furthermore, the molecular basis of how serotonin is recognized and transported remains unclear. In this study, we performed unbiased microsecond-long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational-driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We find that the transition from the occluded to inward-facing state is the rate-limiting step for substrate import and that the substrate decreases the free energy barriers to achieve the inward-facing state. Our study provides insights on the molecular basis of dynamics-driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits.

Class C G protein-coupled receptors (GPCRs) are obligatory dimers that are particularly important for neuronal responses to endogenous and environmental stimuli. Ligand recognition through large extracellular domains leads to the reorganization of transmembrane regions to activate G protein signaling. Although structures of individual domains are known, the complete architecture of a class C GPCR and the mechanism of interdomain coupling during receptor activation are unclear. By screening a mutagenesis library of the human class C sweet taste receptor subunit T1R2, we enhanced surface expression and identified a dibasic intracellular retention motif that modulates surface expression and co-trafficking with its heterodimeric partner T1R3. Using a highly expressed T1R2 variant, dimerization sites along the entire subunit within all the structural domains were identified by a comprehensive mutational scan for co-trafficking with T1R3 in human cells. The data further reveal that the C terminus of the extracellular cysteine-rich domain needs to be properly folded for T1R3 dimerization and co-trafficking, but not for surface expression of T1R2 alone. These results guided the modeling of the T1R2-T1R3 dimer in living cells, which predicts a twisted arrangement of domains around the central axis, and a continuous folded structure between transmembrane domain loops and the cysteine-rich domains. These insights have implications for how conformational changes between domains are coupled within class C GPCRs.

Binding of Sulfoxaflor to Aplysia californica-AChBP: Computational Insights from Multiscale Approaches.

Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.

Universality of the Sodium Ion Binding Mechanism in Class†A G-Protein-Coupled Receptors.

The allosteric modulation of G-protein-coupled receptors (GPCRs) by sodium ions has received significant attention as crystal structures of several receptors show Na+ ions bound to the inactive conformations at the conserved Asp2.50 . To date, structures from 24 families of GPCRs have been determined, though mechanistic insights into Na+ binding to the allosteric site are limited. We performed hundreds-of-microsecond long simulations of 18 GPCRs and elucidated their Na+ binding mechanism. In class A GPCRs, the Na+ ion binds to the conserved residue 2.50 whereas in class B receptors, it binds at 3.43b, 6.53b, and 7.49b. Using Markov state models, we obtained the free energy profiles and kinetics of Na+ binding to the allosteric site, which reveal a conserved mechanism of Na+ binding for GPCRs and show the residues that act as major barriers for ion diffusion. Furthermore, we also show that the Na+ ion can bind to GPCRs from the intracellular side when the allosteric site is inaccessible from the extracellular side.

Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation.

The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein (Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI-Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI-Ac-AChBP amino acid pairs.

Energetics of substrate transport in proton-dependent oligopeptide transporters.

The PepT So transporter mediates the transport of peptides across biological membranes. Despite advancements in structural biology, including cryogenic electron microscopy structures resolving PepT So in different states, the molecular basis of peptide recognition and transport by PepT So is not fully elucidated. In this study, we employed molecular dynamics simulations, Markov State Models (MSMs), and Transition Path Theory (TPT) to investigate the transport mechanism of an alanine-alanine peptide (Ala-Ala) through the PepT So transporter. Our simulations revealed conformational changes and key intermediate states involved in peptide translocation. We observed that the presence of the Ala-Ala peptide substrate lowers the free energy barriers associated with transition to the inward-facing state. Furthermore, we elucidated the proton transport model and analyzed the pharmacophore features of intermediate states, providing insights for rational drug design. These findings highlight the significance of substrate binding in modulating the conformational dynamics of PepT So and identify critical residues that facilitate transport.

SWEET family transporters act as water conducting carrier proteins in plants.

Dedicated water channels are involved in the facilitated diffusion of water molecules across the cell membrane in plants. Transporter proteins are also known to transport water molecules along with substrates, however the molecular mechanism of water permeation is not well understood in plant transporters. Here, we show plant sugar transporters from the SWEET (Sugar Will Eventually be Exported Transporter) family act as water-conducting carrier proteins via a variety of passive and active mechanisms that allow diffusion of water molecules from one side of the membrane to the other. This study provides a molecular perspective on how plant membrane transporters act as water carrier proteins, a topic that has not been extensively explored in literature. Water permeation in membrane transporters could occur via four distinct mechanisms which form our hypothesis for water transport in SWEETs. These hypothesis are tested using molecular dynamics simulations of the outward-facing, occluded, and inward-facing state of AtSWEET1 to identify the water permeation pathways and the flux associated with them. The hydrophobic gates at the center of the transport tunnel act as a barrier that restricts water permeation. We have performed in silico single and double mutations of the hydrophobic gate residues to examine the changes in the water conductivity. Surprisingly, the double mutant allows the water permeation to the intracellular half of the membrane and forms a continuous water channel. These computational results are validated by experimentally examining the transport of hydrogen peroxide molecules by the AtSWEET family of transporters. We have also shown that the transport of hydrogen peroxide follows the similar mechanism as water transport in AtSWEET1. Finally, we conclude that similar water-conduction states are also present in other SWEET transporters due to the high sequence and structure conservation exhibited by this transporter family.

Corrigendum: Cloning and expression of cockroach α7 nicotinic acetylcholine receptor subunit.

[This corrects the article DOI: 10.3389/fphys.2020.00418.].

Simulations of biased agonists in the ß(2) adrenergic receptor with accelerated molecular dynamics.

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though ß-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and ß-arrestin. Here we report on the dynamics of the ß2 adrenergic receptor bound to the ß-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the ß-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs.

Addressing selective polypharmacology of antipsychotic drugs targeting the bioaminergic receptors through receptor dynamic conformational ensembles.

Selective polypharmacology, where a drug acts on multiple rather than a single molecular target involved in a disease, emerges to develop a structure-based system biology approach to design drugs selectively targeting a disease-active protein network. We focus on the bioaminergic receptors that belong to the group of G-protein-coupled receptors (GPCRs) and represent targets for therapeutic agents against schizophrenia and depression. Among them, it has been shown that the serotonin (5-HT(2A) and 5-HT6) and dopamine (D2 and D3) receptors induce a cognition-enhancing effect (group 1), while the histamine (H1) and serotonin (5-HT(2C)) receptors lead to metabolic side effects and the 5-HT(2B) serotonin receptor causes pulmonary hypertension (group 2). Thus, the problem arises to develop an approach that allows identifying drugs targeting only the disease-active receptors, i.e. group 1. The recent release of several crystal structures of the bioaminergic receptors, involving the D3 and H1 receptors, provides the possibility to model the structures of all receptors and initiate a study of the structural and dynamic context of selective polypharmacology. In this work, we use molecular dynamics simulations to generate a conformational space of the receptors and subsequently characterize its binding properties applying molecular probe mapping. All-against-all comparison of the generated probe maps of the selected diverse conformations of all receptors with the Tanimoto similarity coefficient (Tc) enable the separation of the receptors of group 1 from group 2. The pharmacophore built based on the Tc-selected receptor conformations, using the multiple probe maps discovers structural features that can be used to design molecules selective toward the receptors of group 1. The importance of several predicted residues to ligand selectivity is supported by the available mutagenesis and ligand structure-activity relationship studies. In addition, the Tc-selected conformations of the receptors for group 1 show good performance in isolation of known ligands from a random decoy. Our computational structure-based protocol to tackle selective polypharmacology of antipsychotic drugs could be applied for other diseases involving multiple drug targets, such as oncologic and infectious disorders.

Distinct Binding Mechanisms for Allosteric Sodium Ion in Cannabinoid Receptors.

The therapeutic potential of cannabinoid receptors is not fully explored due to psychoactive side effects and lack of selectivity associated with orthosteric ligands. Allosteric modulators have the potential to become selective therapeutics for cannabinoid receptors. Biochemical experiments have shown the effects of the allosteric Na+ binding on cannabinoid receptor activity. However, the Na+ coordination site and binding pathway are still unknown. Here, we perform molecular dynamic simulations to explore Na+ binding in the cannabinoid receptors, CB1 and CB2. Simulations reveal that Na+ binds to the primary binding site from different extracellular sites for CB1 and CB2. A distinct secondary Na+ coordination site is identified in CB1 that is not present in CB2. Furthermore, simulations also show that intracellular Na+ could bind to the Na+ binding site in CB1. Constructed Markov state models show that the standard free energy of Na+ binding is similar to the previously calculated free energy for other class A GPCRs.

How do antiporters exchange substrates across the cell membrane? An atomic-level description of the complete exchange cycle in NarK.

Major facilitator superfamily (MFS) proteins operate via three different mechanisms: uniport, symport, and antiport. Despite extensive investigations, the molecular understanding of antiporters is less advanced than that of other transporters due to the complex coupling between two substrates and the lack of distinct structures. We employ extensive all-atom molecular dynamics simulations to dissect the complete substrate exchange cycle of the bacterial NO3-/NO2- antiporter, NarK. We show that paired basic residues in the binding site prevent the closure of unbound protein and ensure the exchange of two substrates. Conformational transition occurs only in the presence of substrate, which weakens the electrostatic repulsion and stabilizes the transporter. Furthermore, we propose a state-dependent substrate exchange model, in which the relative spacing between the paired basic residues determines whether NO3- and NO2- bind simultaneously or sequentially. Overall, this work presents a general working model for the antiport mechanism within the MFS.

Identification and analysis of sugar transporters capable of co-transporting glucose and xylose simultaneously.

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

Cloning and Expression of Cockroach α7 Nicotinic Acetylcholine Receptor Subunit.

Understanding insect nicotinic acetylcholine receptor (nAChR) subtypes is of major interest because they are the main target of several insecticides. In this study, we have cloned a cockroach Pameα7 subunit that encodes a 518 amino acid protein with futures typical of nAChR subunit, and sequence homology to α7 subunit. Pameα7 is differently expressed in the cockroach nervous system, in particular in the antennal lobes, optical lobes and the mushroom bodies where specific expression was found in the non-compact Kenyon cells. In addition, we found that cockroach Pameα7 subunits expressed in Xenopus laevis oocytes can assemble to form homomeric receptors. Electrophysiological recordings using the two-electrode voltage clamp method demonstrated that nicotine induced an I max current of -92 ± 27 nA at 1 mM. Despite that currents are low with the endogenous ligand, ACh, this study provides information on the first expression of cockroach α7 homomeric receptor.

Distinct Substrate Transport Mechanism Identified in Homologous Sugar Transporters.

SWEETs and their prokaryotic counterparts SemiSWEETs were recently classified as transporters that translocate sugar across cellular membranes. SemiSWEETs are commonly used as a model system to infer biological properties of SWEETs; however, this presumes that the homologues are comparable to begin with. We evaluate this presumption by comparing their protein dynamics and substrate transport mechanism using 532 µs of simulation data in conjunction with Markov state models (MSMs). MSM weighted conformational landscape plots reveal significant differences between SWEETs and SemiSWEETs despite having similar structural topology. The presence of glucose reduces the free energy barrier between the functionally important intermediate states to enhance the transport process, while the substrate has no effect on SemiSWEET. The glucose adopts more rotational degrees of freedom in SWEET, while its conformation is restricted for SemiSWEET. Our study provides biological insights on the unexplored novelty of difference in the functional mechanism of two close homologous proteins.

Molecular Basis of the Glucose Transport Mechanism in Plants.

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane (SWEET, Sugars Will Eventually Be Exported Transporter). However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in the OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as for the glucose transport mechanism. SWEETs undergo a structural transition to outward-facing (OF), occluded (OC), and inward-facing (IF) and strongly support an alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly the same for OF, OC, and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and acts as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass-like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

Free Energy Landscape of the Complete Transport Cycle in a Key Bacterial Transporter.

PepTSo is a proton-coupled bacterial symporter, from the major facilitator superfamily (MFS), which transports di-/tripeptide molecules. The recently obtained crystal structure of PepTSo provides an unprecedented opportunity to gain an understanding of functional insights of the substrate transport mechanism. Binding of the proton and peptide molecule induces conformational changes into occluded (OC) and outward-facing (OF) states, which we are able to characterize using molecular dynamics (MD) simulations. The structural knowledge of the OC and OF state is important to fully understand the major energy barrier associated with the transport cycle. In order to gain functional insight into the interstate dynamics, we performed extensive all atom MD simulations. The Markov state model was constructed to identify the free energy barriers between the states, and kinetic information on intermediate pathways was obtained using the transition pathway theory (TPT). TPT shows that the OF state is obtained by the movement of TM1 and TM7 at the extracellular side approximately 12-16 Å away from each other, and the inward movement of TM4 and TM10 at the intracellular halves to 3-4 Å characterizes the OC state. Helix distance distributions obtained from MD simulations were compared with experimental double electron-electron resonance spectroscopy and were found to be in excellent agreement with previous studies. We also predicted the optimal positions for placement of methane thiosulfonate spin label probes to capture the slowest protein dynamics. Our finding sheds light on the conformational cycle of this key membrane transporter and the functional relationships between the multiple intermediate states.