Mechanism of gating and partial agonist action in the glycine receptor

Mapping Intimacies ◽

10.1101/786632 ◽

2019 ◽

Cited By ~ 2

Author(s):

Jie Yu ◽

Hongtao Zhu ◽

Remigijus Lape ◽

Timo Greiner ◽

Rezvan Shahoei ◽

...

Keyword(s):

Conformational Changes ◽

Molecular Mechanisms ◽

Open Channel ◽

Partial Agonist ◽

Glycine Receptor ◽

Md Simulations ◽

Binding Pocket ◽

Closed Channel ◽

Agonist Action ◽

Receptor Family

SummaryThe glycine receptor is a pentameric, neurotransmitter-activated ion channel that transitions between closed/resting, open and desensitized states. Glycine, a full agonist, produces an open channel probability (Po) of ∼1.0 while partial agonists, such as taurine and γ-amino butyric acid (GABA) yield submaximal Po values. Despite extensive studies of pentameric Cys-loop receptors, there is little knowledge of the molecular mechanisms underpinning partial agonist action and how the receptor transitions from the closed to open and to desensitized conformations. Here we use electrophysiology and molecular dynamics (MD) simulations, together with a large ensemble of single particle cryo-EM reconstructions, to show how agonists populate agonist-bound yet closed channel states, thus explaining their lesser efficacy, yet also populate agonist-bound open and desensitized states. Measurements within the neurotransmitter binding pocket, as a function of bound agonist, provide a metric to correlate the extent of agonist-induced conformational changes to open channel probability across the Cys-loop receptor family.

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m400548200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19559-19565 ◽

Cited By ~ 14

Author(s):

Nian-Lin R. Han ◽

John D. Clements ◽

Joseph W. Lynch

Keyword(s):

Conformational Changes ◽

Reaction Rate ◽

Partial Agonist ◽

Glycine Receptor ◽

Reaction Rates ◽

Structural Basis ◽

Surface Accessibility ◽

Partial Agonists ◽

Substituted Cysteine Accessibility ◽

Receptor Family

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg271to Lys276in the M2-M3 domain of the homomeric α1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT3Aserotonin receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908848117 ◽

2019 ◽

Vol 117 (1) ◽

pp. 405-414 ◽

Cited By ~ 8

Author(s):

Nicholas B. Guros ◽

Arvind Balijepalli ◽

Jeffery B. Klauda

Keyword(s):

Serotonin Receptor ◽

Molecular Mechanisms ◽

Transmembrane Domain ◽

Allosteric Regulation ◽

Md Simulations ◽

Binding Pocket ◽

Careful Examination ◽

Dynamic Binding ◽

Order Of Magnitude ◽

Membrane Bound

Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure–function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT3A) serotonin receptor for 15 to 20 μs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT3A, allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT3A, the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation.

Insights into the substrate binding mechanism of SULT1A1 through molecular dynamics with excited normal modes simulations

Scientific Reports ◽

10.1038/s41598-021-92480-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Balint Dudas ◽

Daniel Toth ◽

David Perahia ◽

Arnaud B. Nicot ◽

Erika Balog ◽

...

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Molecular Mechanisms ◽

Normal Modes ◽

Md Simulations ◽

Conformational Space ◽

Metabolizing Enzymes ◽

Protein Ligand Interactions ◽

Ligand Interactions ◽

Excited Normal Modes

AbstractSulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein–ligand interactions studies where major rearrangements are expected.

Structural basis for σ1 receptor ligand recognition

10.1101/333765 ◽

2018 ◽

Author(s):

Hayden R. Schmidt ◽

Robin M. Betz ◽

Ron O. Dror ◽

Andrew C. Kruse

Keyword(s):

Ligand Binding ◽

Conformational Changes ◽

Md Simulations ◽

Integral Membrane Protein ◽

Structural Basis ◽

Σ1 Receptor ◽

Agonist Action ◽

Slow Kinetics ◽

Multistep Process ◽

G Protein Coupled

The σ1 receptor is a poorly understood integral membrane protein expressed in most cells and tissues in the human body. It has been shown to modulate the activity of other membrane proteins such as ion channels and G protein-coupled receptors1–4, and ligands targeting the σ1 receptor are currently in clinical trials for treatment of Alzheimer’s disease5, ischemic stroke6, and neuropathic pain7. Despite its importance, relatively little is known regarding σ1 receptor function at the molecular level. Here, we present crystal structures of the human σ1 receptor bound to the classical antagonists haloperidol and NE-100, as well as the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 Å, 2.9 Å, and 3.1 Å respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations demonstrate that the agonist induces subtle structural rearrangements in the receptor. In addition, we show that ligand binding and dissociation from σ1 is a multistep process, with extraordinarily slow kinetics limited by receptor conformational change. We use MD simulations to reconstruct a ligand binding pathway that requires two major conformational changes. Taken together, these data provide a framework for understanding the molecular basis for agonist action at σ1.

Ligand Binding Mechanism and Its Relationship with Conformational Changes in Adenine Riboswitch

International Journal of Molecular Sciences ◽

10.3390/ijms21061926 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1926

Author(s):

Guodong Hu ◽

Haiyan Li ◽

Shicai Xu ◽

Jihua Wang

Keyword(s):

Ligand Binding ◽

Conformational Change ◽

Conformational Changes ◽

Md Simulations ◽

Binding Pocket ◽

Dynamical Analysis ◽

Mass Center ◽

Free Energies ◽

Binding Model ◽

Binding Free Energies

Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics and chemical tools. The adenine riboswitch can bind not only to purine analogues but also to pyrimidine analogues. Here, long molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) computational methodologies were carried out to show the differences in the binding model and the conformational changes upon five ligands binding. The binding free energies of the guanine riboswitch aptamer with C74U mutation complexes were compared to the binding free energies of the adenine riboswitch (AR) aptamer complexes. The calculated results are in agreement with the experimental data. The differences for the same ligand binding to two different aptamers are related to the electrostatic contribution. Binding dynamical analysis suggests a flexible binding pocket for the pyrimidine ligand in comparison with the purine ligand. The 18 μs of MD simulations in total indicate that both ligand-unbound and ligand-bound aptamers transfer their conformation between open and closed states. The ligand binding obviously affects the conformational change. The conformational states of the aptamer are associated with the distance between the mass center of two key nucleotides (U51 and A52) and the mass center of the other two key nucleotides (C74 and C75). The results suggest that the dynamical character of the binding pocket would affect its biofunction. To design new ligands of the adenine riboswitch, it is recommended to consider the binding affinities of the ligand and the conformational change of the ligand binding pocket.

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

Biochemical Journal ◽

10.1042/bj20060943 ◽

2007 ◽

Vol 402 (1) ◽

pp. 51-61 ◽

Cited By ~ 13

Author(s):

Brian J. Holleran ◽

Marie-Eve Beaulieu ◽

Christophe D. Proulx ◽

Pierre Lavigne ◽

Emanuel Escher ◽

...

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Binding Sites ◽

Transmembrane Domain ◽

Partial Agonist ◽

Binding Pocket ◽

Activation Mechanism ◽

Urotensin Ii ◽

Agonist Binding ◽

Partial Agonists

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3–17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

Origins of stereoselectivity in evolved ketoreductases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507910112 ◽

2015 ◽

Vol 112 (51) ◽

pp. E7065-E7072 ◽

Cited By ~ 50

Author(s):

Elizabeth L. Noey ◽

Nidhi Tibrewal ◽

Gonzalo Jiménez-Osés ◽

Sílvia Osuna ◽

Jiyong Park ◽

...

Keyword(s):

Conformational Changes ◽

Large Scale ◽

Kinetic Studies ◽

Md Simulations ◽

Binding Pocket ◽

Scale Production ◽

Large Scale Production ◽

Conformational Preferences ◽

Short Chain Alcohol ◽

The Stability

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP+-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Different conformational responses of the β2-adrenergic receptor-Gs complex upon binding of the partial agonist salbutamol or the full agonist isoprenaline

National Science Review ◽

10.1093/nsr/nwaa284 ◽

2020 ◽

Author(s):

Fan Yang ◽

Shenglong Ling ◽

Yingxin Zhou ◽

Yanan Zhang ◽

Pei Lv ◽

...

Keyword(s):

Conformational Changes ◽

Hydrophobic Interactions ◽

Partial Agonist ◽

Binding Pocket ◽

Structural Basis ◽

Spectroscopic Techniques ◽

Toggle Switch ◽

Intracellular Loop ◽

Full Agonist ◽

Β2 Adrenergic Receptor

Abstract GPCRs are responsible for most cytoplasmic signaling in response to extracellular ligands with different efficacy profiles. Various spectroscopic techniques have identified that agonists exhibiting varying efficacies can selectively stabilize a specific conformation of the receptor. However, the structural basis for activation of the GPCR-G protein complex by ligands with different efficacies is incompletely understood. To better understand the structural basis underlying the mechanisms by which ligands with varying efficacies differentially regulate the conformations of receptors and G proteins, we determined the structures of β2AR-Gαs$\beta $γ bound with partial agonist salbutamol or bound with full agonist isoprenaline using single-particle cryo-electron microscopy at resolutions of 3.26 Å and 3.80 Å, respectively. Structural comparisons between the β2AR-Gs-salbutamol and β2AR-Gs-isoprenaline complexes demonstrated that the decreased binding affinity and efficacy of salbutamol compared with those of isoprenaline might be attributed to the weakened hydrogen bonding interactions, attenuated hydrophobic interactions in the orthosteric binding pocket and different conformational changes in the rotamer toggle switch in TM6. Moreover, the observed stronger interactions between the intracellular loop 2 or 3 (ICL2 or ICL3) of β2AR and Gαs with the binding of salbutamol versus isoprenaline might decrease phosphorylation in the salbutamol-activated β2AR-Gs complex. From the observed structural differences between these complexes of β2AR, a mechanism of β2AR activation by partial and full agonists is proposed to shed structural insights for β2AR desensitization.

Structure–Function Relationships in the Human P-glycoprotein (ABCB1): Insights from Molecular Dynamics Simulations

International Journal of Molecular Sciences ◽

10.3390/ijms23010362 ◽

2021 ◽

Vol 23 (1) ◽

pp. 362

Author(s):

Liadys Mora Lagares ◽

Yunierkis Pérez-Castillo ◽

Nikola Minovski ◽

Marjana Novič

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Efflux Pump ◽

Transmembrane Protein ◽

Conformational Dynamics ◽

Water Environment ◽

Md Simulations ◽

Binding Pocket ◽

Target Cells ◽

P Glycoprotein

P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette superfamily of transporters, and it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping compounds out of cells. P-gp contributes to a reduction in toxicity, and has broad substrate specificity. It is involved in the failure of many cancer and antiviral chemotherapies due to the phenomenon of multidrug resistance (MDR), in which the membrane transporter removes chemotherapeutic drugs from target cells. Understanding the details of the ligand–P-gp interaction is therefore critical for the development of drugs that can overcome the MDR phenomenon, for the early identification of P-gp substrates that will help us to obtain a more effective prediction of toxicity, and for the subsequent outdesign of substrate properties if needed. In this work, a series of molecular dynamics (MD) simulations of human P-gp (hP-gp) in an explicit membrane-and-water environment were performed to investigate the effects of binding different compounds on the conformational dynamics of P-gp. The results revealed significant differences in the behaviour of P-gp in the presence of active and non-active compounds within the binding pocket, as different patterns of movement were identified that could be correlated with conformational changes leading to the activation of the translocation mechanism. The predicted ligand–P-gp interactions are in good agreement with the available experimental data, as well as the estimation of the binding-free energies of the studied complexes, demonstrating the validity of the results derived from the MD simulations.