Binding of Azobenzene and p-Diaminoazobenzene to the Human Voltage-Gated Sodium Channel NaV1.4

The activity of voltage-gated ion channels can be controlled by the binding of photoswitches inside their internal cavity and subsequent light irradiation. We investigated the binding of azobenzene and p-diaminoazobenzene to the human Na<sub>V</sub>1.4 channel in the inactivated state by means of Gaussian accelerated molecular dynamics simulations and free-energy computations. Three stable binding pockets were identified for each of the two photoswitches. In all the cases, the binding is controlled by the balance between the favorable hydrophobic interactions of the ligands with the nonpolar residues of the protein and the unfavorable polar solvation energy. In addition, electrostatic interactions between the ligand and the polar aminoacids are also relevant for p-diaminoazobenzene due to the presence of the amino groups on the benzene moieties. These groups participate in hydrogen bonding in the most favorable binding pocket and in long-range electrostatic interactions in the other pockets. The thermodinamically preferred binding sites found for both photoswitches are close to the selectivity filter of the channel. Therefore, it is very likely that the binding of these ligands will induce alterations in the ion conduction through the channel.

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Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1905462116 ◽

2019 ◽

Vol 116 (38) ◽

pp. 18951-18961 ◽

Cited By ~ 6

Author(s):

Richard Banh ◽

Vladimir V. Cherny ◽

Deri Morgan ◽

Boris Musset ◽

Sarah Thomas ◽

...

Keyword(s):

Proton Channel ◽

Voltage Gated ◽

Closed State ◽

Channel Opening ◽

Proton Current ◽

Voltage Dependent ◽

Current Flows ◽

Voltage Gated Ion Channels ◽

Dynamics Simulations ◽

Voltage Sensing Domain

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

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A novel Hv1 inhibitor reveals a new mechanism of inhibition of a voltage-sensing domain

The Journal of General Physiology ◽

10.1085/jgp.202012833 ◽

2021 ◽

Vol 153 (9) ◽

Author(s):

Chang Zhao ◽

Liang Hong ◽

Saleh Riahi ◽

Victoria T. Lim ◽

Douglas J. Tobias ◽

...

Keyword(s):

Md Simulations ◽

Binding Pocket ◽

Proton Channel ◽

Voltage Sensing ◽

Voltage Gated ◽

Functional Studies ◽

Mechanism Of Inhibition ◽

Target Binding ◽

Voltage Gated Ion Channels ◽

Pore Domain

Voltage-gated sodium, potassium, and calcium channels consist of four voltage-sensing domains (VSDs) that surround a central pore domain and transition from a down state to an up state in response to membrane depolarization. While many types of drugs bind pore domains, the number of organic molecules known to bind VSDs is limited. The Hv1 voltage-gated proton channel is made of two VSDs and does not contain a pore domain, providing a simplified model for studying how small ligands interact with VSDs. Here, we describe a ligand, named HIF, that interacts with the Hv1 VSD in the up and down states. We find that HIF rapidly inhibits proton conduction in the up state by blocking the open channel, as previously described for 2-guanidinobenzimidazole and its derivatives. HIF, however, interacts with a site slowly accessible in the down state. Functional studies and MD simulations suggest that this interaction traps the compound in a narrow pocket lined with charged residues within the VSD intracellular vestibule, which results in slow recovery from inhibition. Our findings point to a “wrench in gears” mechanism whereby side chains within the binding pocket trap the compound as the teeth of interlocking gears. We propose that the use of screening strategies designed to target binding sites with slow accessibility, similar to the one identified here, could lead to the discovery of new ligands capable of interacting with VSDs of other voltage-gated ion channels in the down state.

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The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase

eLife ◽

10.7554/elife.41653 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 4

Author(s):

Akira Kawanabe ◽

Masaki Hashimoto ◽

Manami Nishizawa ◽

Kazuhisa Nishizawa ◽

Hirotaka Narita ◽

...

Keyword(s):

Conformational Changes ◽

Membrane Association ◽

Membrane Interface ◽

Voltage Sensing ◽

Phosphatase Domain ◽

Voltage Gated ◽

Voltage Dependent ◽

Hydrophobic Nature ◽

Voltage Gated Ion Channels ◽

Dynamics Simulations

Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive. Here we report a membrane interface (named ‘the hydrophobic spine’), which is essential for the coupling of the VSD and CCR. Our molecular dynamics simulations suggest that the hydrophobic spine of Ciona intestinalis VSP (Ci-VSP) provides a hinge-like motion for the CCR through the loose membrane association of the phosphatase domain. Electrophysiological experiments indicate that the voltage-dependent phosphatase activity of Ci-VSP depends on the hydrophobicity and presence of an aromatic ring in the hydrophobic spine. Analysis of conformational changes in the VSD and CCR suggests that the VSP has two states with distinct enzyme activities and that the second transition depends on the hydrophobic spine.

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Dynamic Control of Ca2+ Binding in the C2 Domains of Synaptotagmin 1

10.1101/412031 ◽

2018 ◽

Author(s):

Patrick J. Rock ◽

Austin G. Meyer ◽

Chantell S. Evans ◽

Edwin R. Chapman ◽

R. Bryan Sutton

Keyword(s):

Structural Changes ◽

Dynamic Control ◽

Point Mutations ◽

Binding Pocket ◽

Snare Complex ◽

C2 Domains ◽

Domain Interactions ◽

Synaptotagmin 1 ◽

Binding Pockets ◽

Dynamics Simulations

AbstractSynaptotagmin senses fluctuations in the Ca2+ environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+ at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+ -binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+ binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+ -binding pockets of C2A and C2B. The volume of the unbound Ca2+ -binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+ -binding pocket in C2A. C2B maintains a more compact Ca2+ -binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+ binding loops would likely affect Ca2+ affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+ -binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+ -binding pocket and provides an explanation for the observed lethal phenotype.

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In-Silico Electrophysiology: On the Activation of Voltage-Gated Ion Channels using Molecular Dynamics Simulations

Biophysical Journal ◽

10.1016/j.bpj.2015.11.633 ◽

2016 ◽

Vol 110 (3) ◽

pp. 107a ◽

Cited By ~ 1

Author(s):

Mounir Tarek ◽

Lucie Delemotte ◽

Marina Kasimova ◽

Michael L. Klein ◽

Vincenzo Carnevale

Keyword(s):

Molecular Dynamics ◽

Ion Channels ◽

Molecular Dynamics Simulations ◽

In Silico ◽

Voltage Gated ◽

Voltage Gated Ion Channels ◽

Dynamics Simulations ◽

Gated Ion Channels

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Mutation of Tyr137 of the universalEscherichia colifimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding

IUCrJ ◽

10.1107/s2052252516016675 ◽

2017 ◽

Vol 4 (1) ◽

pp. 7-23 ◽

Cited By ~ 13

Author(s):

Said Rabbani ◽

Eva-Maria Krammer ◽

Goedele Roos ◽

Adam Zalewski ◽

Roland Preston ◽

...

Keyword(s):

Ethylenediaminetetraacetic Acid ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Lectin Domain ◽

E Coli ◽

Ligand Free ◽

Mannose Binding ◽

Bowel Diseases ◽

Inflammatory Bowel ◽

Dynamics Simulations

The most prevalent diseases manifested byEscherichia coliare acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease.E. coliclinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connectedviaa pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α-D-O-mannopyranoside or 4-biphenyl α-D-O-mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α-D-O-mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48viathe interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant.

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Peptide Terminus Tilting: an Unusual conformational transition of MHC Class I Revealed by Molecular Dynamics Simulations

10.1101/219873 ◽

2017 ◽

Author(s):

Yeping Sun ◽

Po Tian

Keyword(s):

Molecular Dynamics ◽

Antigen Presentation ◽

Influenza A ◽

Conformational Transition ◽

Md Simulations ◽

Lymphocytic Choriomeningitis ◽

Class I ◽

Accelerated Molecular Dynamics ◽

Binding Pockets ◽

Dynamics Simulations

ABSTRACTA conventional picture for major histocompatibility complex class I (MHCI) antigen presentation is that the terminal anchor residues of the antigenic peptide bind to the pockets at the bottom of the MHC cleft, leaving the central peptide residues exposed for T cell antigen receptor (TCR) recognition. However, in the present study, we show that in canonical or accelerated molecular dynamics (MD) simulations, the peptide terminus in some immunodominant peptide-MHCI (pMHCI) complexes can detach from their binding pockets and stretch outside the MHC cleft. These pMHCI complexes include the complex of the H-2Kb and the lymphocytic choriomeningitis virus (LCMV) gp33 peptide, and the complex of the HLA-A*0201 and the influenza A virus M1 peptide. The detached peptide terminus becomes the most prominent spot at the pMHC interface, and so can serves as a novel TCR recognition target. Thus, peptide terminus detaching may be a novel mechanism for MHC antigen presentation.

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Enhancing Sidechain Rotamer Sampling Using Non-Equilibrium Candidate Monte Carlo

10.26434/chemrxiv.7184876 ◽

2019 ◽

Author(s):

Kalistyn H. Burley ◽

Samuel C. Gill ◽

Nathan M. Lim ◽

David Mobley

Keyword(s):

Molecular Dynamics ◽

Monte Carlo ◽

Ligand Binding ◽

Electrostatic Interactions ◽

Molecular Simulations ◽

Adequate Sampling ◽

Binding Pockets ◽

Sidechain Rotamers ◽

Dynamics Simulations ◽

Non Equilibrium

<div>Molecular simulations are a valuable tool for studying biomolecular motions and thermodynamics. However, such motions can be slow compared to simulation timescales, yet critical. Specifically, adequate sampling of sidechain motions in protein binding pockets proves crucial for obtaining accurate estimates of ligand binding free energies from molecular simulations. The timescale of sidechain rotamer flips can range from a few ps to several hundred ns or longer, particularly in crowded environments like the interior of proteins. Here, we apply a mixed non-equilibrium candidate Monte Carlo (NCMC)/molecular dynamics (MD) method to enhance sampling of sidechain rotamers. The NCMC portion of our method applies a switching protocol wherein the steric and electrostatic interactions between target sidechain atoms and the surrounding environment are cycled off and then back on during the course of a move proposal. Between NCMC move proposals, simulation of the system continues via traditional molecular dynamics. Here, we first validate this approach on a simple, solvated valine-alanine dipeptide system and then apply it to a well-studied model ligand binding site in T4 lysozyme L99A. We compute the rate of rotamer transitions for a valine sidechain using our approach and compare it to that of traditional molecular dynamics simulations. Here, we show that our NCMC/MD method substantially enhances sidechain sampling, especially in systems where the torsional barrier to rotation is high (>10 kcal/mol). These barriers can be intrinsic torsional barriers or steric barriers imposed by the environment.</div><div>Overall, this may provide a promising strategy to selectively improve sidechain sampling in molecular simulations.</div>

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Enhancing Sidechain Rotamer Sampling Using Non-Equilibrium Candidate Monte Carlo

10.26434/chemrxiv.7184876.v1 ◽

2018 ◽

Author(s):

Kalistyn H. Burley ◽

Samuel C. Gill ◽

Nathan M. Lim ◽

David Mobley

Keyword(s):

Molecular Dynamics ◽

Monte Carlo ◽

Ligand Binding ◽

Electrostatic Interactions ◽

Molecular Simulations ◽

Adequate Sampling ◽

Binding Pockets ◽

Sidechain Rotamers ◽

Dynamics Simulations ◽

Non Equilibrium

<div>Molecular simulations are a valuable tool for studying biomolecular motions and thermodynamics. However, such motions can be slow compared to simulation timescales, yet critical. Specifically, adequate sampling of sidechain motions in protein binding pockets proves crucial for obtaining accurate estimates of ligand binding free energies from molecular simulations. The timescale of sidechain rotamer flips can range from a few ps to several hundred ns or longer, particularly in crowded environments like the interior of proteins. Here, we apply a mixed non-equilibrium candidate Monte Carlo (NCMC)/molecular dynamics (MD) method to enhance sampling of sidechain rotamers. The NCMC portion of our method applies a switching protocol wherein the steric and electrostatic interactions between target sidechain atoms and the surrounding environment are cycled off and then back on during the course of a move proposal. Between NCMC move proposals, simulation of the system continues via traditional molecular dynamics. Here, we first validate this approach on a simple, solvated valine-alanine dipeptide system and then apply it to a well-studied model ligand binding site in T4 lysozyme L99A. We compute the rate of rotamer transitions for a valine sidechain using our approach and compare it to that of traditional molecular dynamics simulations. Here, we show that our NCMC/MD method substantially enhances sidechain sampling, especially in systems where the torsional barrier to rotation is high (>10 kcal/mol). These barriers can be intrinsic torsional barriers or steric barriers imposed by the environment.</div><div>Overall, this may provide a promising strategy to selectively improve sidechain sampling in molecular simulations.</div>

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