Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage

AbstractThe connexin family is a diverse group of highly regulated non-β-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3’,5’-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 μs simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 μs of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner.Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-β-barrel wide-pore channels.

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Exploring the Binding Interaction of Raf Kinase Inhibitory Protein With the N-Terminal of C-Raf Through Molecular Docking and Molecular Dynamics Simulation

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.655035 ◽

2021 ◽

Vol 8 ◽

Author(s):

Shraddha Parate ◽

Shailima Rampogu ◽

Gihwan Lee ◽

Jong Chan Hong ◽

Keun Woo Lee

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Binding Sites ◽

Binding Free Energy ◽

Md Simulations ◽

Dynamics Simulation ◽

Web Servers ◽

Inhibitory Protein ◽

Raf Kinase ◽

Raf Kinase Inhibitory Protein

Protein-protein interactions are indispensable physiological processes regulating several biological functions. Despite the availability of structural information on protein-protein complexes, deciphering their complex topology remains an outstanding challenge. Raf kinase inhibitory protein (RKIP) has gained substantial attention as a favorable molecular target for numerous pathologies including cancer and Alzheimer’s disease. RKIP interferes with the RAF/MEK/ERK signaling cascade by endogenously binding with C-Raf (Raf-1 kinase) and preventing its activation. In the current investigation, the binding of RKIP with C-Raf was explored by knowledge-based protein-protein docking web-servers including HADDOCK and ZDOCK and a consensus binding mode of C-Raf/RKIP structural complex was obtained. Molecular dynamics (MD) simulations were further performed in an explicit solvent to sample the conformations for when RKIP binds to C-Raf. Some of the conserved interface residues were mutated to alanine, phenylalanine and leucine and the impact of mutations was estimated by additional MD simulations and MM/PBSA analysis for the wild-type (WT) and constructed mutant complexes. Substantial decrease in binding free energy was observed for the mutant complexes as compared to the binding free energy of WT C-Raf/RKIP structural complex. Furthermore, a considerable increase in average backbone root mean square deviation and fluctuation was perceived for the mutant complexes. Moreover, per-residue energy contribution analysis of the equilibrated simulation trajectory by HawkDock and ANCHOR web-servers was conducted to characterize the key residues for the complex formation. One residue each from C-Raf (Arg398) and RKIP (Lys80) were identified as the druggable “hot spots” constituting the core of the binding interface and corroborated by additional long-time scale (300 ns) MD simulation of Arg398Ala mutant complex. A notable conformational change in Arg398Ala mutant occurred near the mutation site as compared to the equilibrated C-Raf/RKIP native state conformation and an essential hydrogen bonding interaction was lost. The thirteen binding sites assimilated from the overall analysis were mapped onto the complex as surface and divided into active and allosteric binding sites, depending on their location at the interface. The acquired information on the predicted 3D structural complex and the detected sites aid as promising targets in designing novel inhibitors to block the C-Raf/RKIP interaction.

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Cannabidiol Selectively Binds to the Voltage-Gated Sodium Channel Nav1.4 in Its Slow-Inactivated State and Inhibits Sodium Current

Biomedicines ◽

10.3390/biomedicines9091141 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1141

Author(s):

Chiung-Wei Huang ◽

Pi-Chen Lin ◽

Jian-Lin Chen ◽

Ming-Jen Lee

Keyword(s):

Binding Sites ◽

Sodium Current ◽

Binding Kinetics ◽

Muscle Membrane ◽

Dependent Manner ◽

Differential Inhibition ◽

Fast Inactivation ◽

Cell Recording ◽

Domain I ◽

Kinetics Of

Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.

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Redox-coupled quinone dynamics in the respiratory complex I

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805468115 ◽

2018 ◽

Vol 115 (36) ◽

pp. E8413-E8420 ◽

Cited By ~ 32

Author(s):

Judith Warnau ◽

Vivek Sharma ◽

Ana P. Gamiz-Hernandez ◽

Andrea Di Luca ◽

Outi Haapanen ◽

...

Keyword(s):

Free Energy ◽

Binding Sites ◽

Complex I ◽

Redox State ◽

Effective Diffusion ◽

Proton Pumping ◽

Free Energy Calculations ◽

Dependent Manner ◽

Membrane Domain ◽

State Dependent

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

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Formation Mechanism of Ion Channel in Channelrhodopsin-2: Molecular Dynamics Simulation and Steering Molecular Dynamics Simulations

International Journal of Molecular Sciences ◽

10.3390/ijms20153780 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3780 ◽

Cited By ~ 1

Author(s):

Ting Yang ◽

Wenying Zhang ◽

Jie Cheng ◽

Yanhong Nie ◽

Qi Xin ◽

...

Keyword(s):

Molecular Dynamics ◽

Ion Channel ◽

Molecular Dynamics Simulations ◽

Formation Mechanism ◽

Binding Sites ◽

Electrostatic Interactions ◽

Potential Of Mean Force ◽

Dynamics Simulation ◽

Cationic Channel ◽

Dynamics Simulations

Channelrhodopsin-2 (ChR2) is a light-activated and non-selective cationic channel protein that can be easily expressed in specific neurons to control neuronal activity by light. Although ChR2 has been extensively used as an optogenetic tool in neuroscience research, the molecular mechanism of cation channel formation following retinal photoisomerization in ChR2 is not well understood. In this paper, studies of the closed and opened state ChR2 structures are presented. The formation of the cationic channel is elucidated in atomic detail using molecular dynamics simulations on the all-trans-retinal (ChR2-trans) configuration of ChR2 and its isomerization products, 13-cis-retinal (ChR2-cis) configuration, respectively. Photoisomerization of the retinal-chromophore causes the destruction of interactions among the crucial residues (e.g., E90, E82, N258, and R268) around the channel and the extended H-bond network mediated by numerous water molecules, which opens the pore. Steering molecular dynamics (SMD) simulations show that the electrostatic interactions at the binding sites in intracellular gate (ICG) and central gate (CG) can influence the transmembrane transport of Na+ in ChR2-cis obviously. Potential of mean force (PMF) constructed by SMD and umbrella sampling also found the existing energy wells at these two binding sites during the transportation of Na+. These wells partly hinder the penetration of Na+ into cytoplasm through the ion channel. This investigation provides a theoretical insight on the formation mechanism of ion channels and the mechanism of ion permeation.

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Protamine Neutralization of the Release of Tissue Factor Pathway Inhibitor Activity by Heparins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649704 ◽

1993 ◽

Vol 70 (06) ◽

pp. 0942-0945 ◽

Cited By ~ 18

Author(s):

Job Harenberg ◽

Marietta Siegele ◽

Carl-Erik Dempfle ◽

Gerd Stehle ◽

Dieter L Heene

Keyword(s):

Tissue Factor ◽

Binding Sites ◽

Tissue Factor Pathway Inhibitor ◽

Factor Xa ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Factor X ◽

Rebound Phenomenon ◽

Tissue Factor Pathway ◽

Kinetics Of

SummaryThe present study was designed to investigate the action of protamine on the release of tissue factor pathway inhibitor (TFPI) activity by unfractionated (UF) and low molecular weight (LMW) heparin in healthy individuals. 5000 IU UF-heparin or 5000 IU LMW-heparin were given intravenously followed by saline, 5000 U protamine chloride or 5000 U protamine sulfate intravenously after the 10 min blood sample. Then serial blood samples for the measurement of TFPI activity and anti-factor Xa- activity were taken, in order to detect a possible relation between the remaining anti-factor X a activity after neutralization of LMW-heparin with protamine and TFPI activity and to establish whether or not a rebound phenomenon of plasmatic TFPI occurs.There was no difference in the release and in the kinetics of TFPI by UF- and LMW-heparin with subsequent administration of saline. After administration of protamine TFPI activity decreased immediately and irreversibly to pretreatment values. There were no differences between protamine chloride and protamine sulfate on the effect of TFPI induced by UF- or LMW-heparin. No rebound phenomenon of TFPI activity occurred. In contrast anti-factor Xa- activity, as measured by the chromogenic S2222-assay, issued the known differences between UF- and LMW-heparin. The half-life of the aXa-effect of LMW-heparin was twice as long as of UF-heparin. Protamine antagonized UF-heparin completely and about 60% of the anti-factor Xa activity of LMW-heparin, using chromogenic S2222-method. No differences could be detected for protamine chloride and sulfate form of protamineIt is assumed that protamine displaces heparins from the binding sites of TFPI. There were no differences between UF- and LMW-heparin. The data indicate that the sustained antifactor Xa activity after antagonization of LMW-heparins as well as heparin rebound phenomena are not mediated by TFPI activity.

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Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661099 ◽

1984 ◽

Vol 51 (03) ◽

pp. 349-353 ◽

Cited By ~ 9

Author(s):

C Caranobe ◽

P Sié ◽

F Fernandez ◽

J Pris ◽

S Moatti ◽

...

Keyword(s):

Binding Sites ◽

High Capacity ◽

Specific Inhibitor ◽

Myeloproliferative Disorders ◽

Normal Subjects ◽

Binding Data ◽

Full Explanation ◽

Number Of Binding Sites ◽

5 Ht Uptake ◽

Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

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m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

10.1055/s-0038-1665767 ◽

1979 ◽

Author(s):

D Bing ◽

D Robison ◽

J Andrews ◽

R Laura

Keyword(s):

Binding Sites ◽

Serine Proteases ◽

Enzyme Inhibitor ◽

Molecular Model ◽

Factor Xa ◽

Affinity Labeling ◽

Catalytic Center ◽

Inhibitor Complex ◽

Polypeptide Chains ◽

Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

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Reversibly Sampling Conformations and Binding Modes Using Molecular Darting

10.26434/chemrxiv.12670676.v2 ◽

2020 ◽

Author(s):

Samuel C. Gill ◽

David Mobley

Keyword(s):

Monte Carlo ◽

Ligand Binding ◽

Binding Sites ◽

Degrees Of Freedom ◽

Dynamics Simulation ◽

Hiv Integrase ◽

Binding Modes ◽

System A ◽

Multiple Binding ◽

Internal Degrees Of Freedom

<div>Sampling multiple binding modes of a ligand in a single molecular dynamics simulation is difficult. A given ligand may have many internal degrees of freedom, along with many different ways it might orient itself a binding site or across several binding sites, all of which might be separated by large energy barriers. We have developed a novel Monte Carlo move called Molecular Darting (MolDarting) to reversibly sample between predefined binding modes of a ligand. Here, we couple this with nonequilibrium candidate Monte Carlo (NCMC) to improve acceptance of moves.</div><div>We apply this technique to a simple dipeptide system, a ligand binding to T4 Lysozyme L99A, and ligand binding to HIV integrase in order to test this new method. We observe significant increases in acceptance compared to uniformly sampling the internal, and rotational/translational degrees of freedom in these systems.</div>

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Titration Simulations for Understanding of the Binding Equilibrium

10.26434/chemrxiv.12980348.v1 ◽

2020 ◽

Author(s):

Dae Hyup Sohn

Keyword(s):

Equilibrium Model ◽

Binding Sites ◽

Binding Constants ◽

Reliability Evaluation ◽

Binding Isotherm ◽

Guest Chemistry

<p>The reliability evaluation of the predicted binding constants in numerous models is also a challenge for supramolecular host-guest chemistry. Here, I briefly formulate binding isotherm with the derivation of the multivalent equilibrium model for the chemist who wants to determine the binding constants of their compounds. This article gives an in-depth understanding of the stoichiometry of binding equilibrium to take divalent binding equilibria bearing two structurally identical binding sites as an example. The stoichiometry of binding equilibrium is affected by (1) the cooperativity of complex, (2) the concentration of titration media, and (3) the equivalents of guests. The simulations were conducted with simple Python codes.</p>

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Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

10.26434/chemrxiv.7505330 ◽

2018 ◽

Author(s):

Luke Jordan ◽

Nathan Wittenberg

Keyword(s):

Lipid Bilayers ◽

Binding Kinetics ◽

Supported Lipid Bilayers ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Supported Lipid Bilayer ◽

Head Groups ◽

Lipid Diffusion ◽

Kinetics Of ◽

Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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