Multiple binding sites for resin-acid derivatives on the voltage-sensor domain of the Shaker potassium channel

ABSTRACTNegatively charged resin acids and their derivatives open voltage-gated potassium (KV) channels by attracting the positively charged voltage-sensor helix of the channel (S4) towards the extracellular leaflet of the cellular membrane and thereby favoring gate opening. The resin acids have been proposed to primarily bind in a pocket in the periphery of the channel, located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. However, in apparent contrast to the suggested electrostatic mechanism, neutralization of the top gating charge of the Shaker KV channel did not reduce the resin-acid induced opening, but unexpectedly increased it, suggesting other mechanisms and other sites of action. Here we explored the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated open state Shaker KV channel by a combination of in-silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resinacid binding sites around the voltage sensor helix S4: (1) The S3/S4 site suggested previously. Positively charged residues introduced at the top of S4 are critical to keep the compound bound in this site by electrostatic force. (2) A site located in the cleft between S4 and the pore domain (the S4/pore site). A tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound. (3) A site located at the extracellular side of the voltage-sensor domain in a cleft formed by S1-S4 (the top-VSD site). The presence of multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation makes the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding pose and the types of interactions it can form with the protein in a state-specific manner.

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Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel

The Journal of General Physiology ◽

10.1085/jgp.202012676 ◽

2021 ◽

Vol 153 (4) ◽

Author(s):

Malin Silverå Ejneby ◽

Arina Gromova ◽

Nina E. Ottosson ◽

Stina Borg ◽

Argel Estrada-Mondragón ◽

...

Keyword(s):

Binding Sites ◽

Resin Acid ◽

Voltage Sensor ◽

Resin Acids ◽

Voltage Gated ◽

Kv Channel ◽

Gating Charge ◽

Extracellular Side ◽

A Site ◽

Positively Charged

Voltage-gated potassium (KV) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.

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EVIDENCE FOR MULTIPLE BINDING SITES OF HIRUDIN IN THROMBIN

10.1055/s-0038-1644666 ◽

1987 ◽

Author(s):

German B Villanueva ◽

Konno Sensuke ◽

John Fenton

Keyword(s):

Dissociation Constant ◽

Active Site ◽

Binding Sites ◽

Specific Activity ◽

Multiple Binding Sites ◽

Straight Line ◽

Multiple Binding ◽

Hyperbolic Curve ◽

Sigmoidal Curve ◽

A Site

A highly purified hirudin with a specific activity of 13, 950 AT units/mg was used in these studies. Investigation of the circular dichroism of hirudin and thrombin showed that the CD spectrum of the thrombin-hirudin complex deviates significantly from additivity towards a less organized structure (i.e. loss of a-helix).A sigmoidal curve, rather than a hyperbolic curve, is generated when the deviation from additivity is plotted against hirudin concentration. This suggests cooperativity of the binding process. At low concentation, aScatchard plot of the data fits intoa straight line clearly indicating one binding site per mole of thrombin.This site binds hirudin with a dissociation constant of 500 nM. However, the data cannot be fitted to a straight line at higher concentration ofhirudin suggesting that hirudin binds also to another site with a different affinity. These results agree with the findings of Stone and Hofsteenge (Biochemistry 25, 4622-4628, 1986) and support the idea that initially hirudin binds at a site distinct from the active site, which then rearranges through a conformational change (detected by CD) to form a tigher complex in which hirudin is also bound to the active site.

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Faculty Opinions recommendation of Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5804956.5786054 ◽

2010 ◽

Author(s):

Edward Bertaccini

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Binding Sites ◽

Transmembrane Domain ◽

General Anesthetic ◽

Nicotinic Acetylcholine ◽

Multiple Binding Sites ◽

Multiple Binding

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NMDAR PAMs: Multiple Chemotypes for Multiple Binding Sites

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666191011095341 ◽

2019 ◽

Vol 19 (24) ◽

pp. 2239-2253 ◽

Cited By ~ 2

Author(s):

Paul J. Goldsmith

Keyword(s):

Binding Sites ◽

Receptor Activation ◽

Research Area ◽

Allosteric Modulators ◽

Nonselective Cation Channels ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Excitatory Neurotransmission ◽

Psychiatric Conditions ◽

High Level

The N-methyl-D-aspartate receptor (NMDAR) is a member of the ionotropic glutamate receptor (iGluR) family that plays a crucial role in brain signalling and development. NMDARs are nonselective cation channels that are involved with the propagation of excitatory neurotransmission signals with important effects on synaptic plasticity. NMDARs are functionally and structurally complex receptors, they exist as a family of subtypes each with its own unique pharmacological properties. Their implication in a variety of neurological and psychiatric conditions means they have been a focus of research for many decades. Disruption of NMDAR-related signalling is known to adversely affect higherorder cognitive functions (e.g. learning and memory) and the search for molecules that can recover (or even enhance) receptor output is a current strategy for CNS drug discovery. A number of positive allosteric modulators (PAMs) that specifically attempt to overcome NMDAR hypofunction have been discovered. They include various chemotypes that have been found to bind to several different binding sites within the receptor. The heterogeneity of chemotype, binding site and NMDAR subtype provide a broad landscape of ongoing opportunities to uncover new features of NMDAR pharmacology. Research on NMDARs continues to provide novel mechanistic insights into receptor activation and this review will provide a high-level overview of the research area and discuss the various chemical classes of PAMs discovered so far.

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Effects of Multiple Binding Sites on Studies of Hydrogen Bonding between Nitroxide Radicals and Solvent Molecules

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19930047 ◽

1993 ◽

Vol 58 (1) ◽

pp. 47-52 ◽

Cited By ~ 2

Author(s):

Imad Al-Bala'a ◽

Richard D. Bates

Keyword(s):

Hydrogen Bonding ◽

Magnetic Resonance ◽

Binding Site ◽

Binding Sites ◽

Hyperfine Coupling Constant ◽

Hydrogen Donor ◽

Complex Formation Constants ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Solvent Molecules

The role of more than one binding site on a nitroxide free radical in magnetic resonance determinations of the properties of the complex formed with a hydrogen donor is examined. The expression that relates observed hyperfine couplings in EPR spectra to complex formation constants and concentrations of each species in solution becomes much more complex when multiple binding sites are present, but reduces to a simpler form when binding at the two sites occurs independently and the binding at the non-nitroxide site does not produce significant differences in the hyperfine coupling constant in the complexed radical. Effects on studies of hydrogen bonding between multiple binding site nitroxides and hydrogen donor solvent molecules by other magnetic resonance methods are potentially more extreme.

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