Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

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A Functionally Conserved Mechanism of Modulation via a Vestibule Site in Pentameric Ligand-Gated Ion Channels

10.1101/770719 ◽

2019 ◽

Author(s):

Marijke Brams ◽

Cedric Govaerts ◽

Kumiko Kambara ◽

Kerry Price ◽

Radovan Spurny ◽

...

Keyword(s):

Ion Channels ◽

Binding Site ◽

Binding Sites ◽

Allosteric Modulators ◽

Channel Opening ◽

Single Domain Antibodies ◽

Allosteric Binding Sites ◽

Target Binding ◽

Gated Ion Channels ◽

Ligand Gated Ion Channels

ABSTRACTPentameric ligand-gated ion channels (pLGICs) belong to a class of ion channels involved in fast synaptic signaling in the central and peripheral nervous systems. Molecules acting as allosteric modulators target binding sites that are remote from the neurotransmitter binding site, but functionally affect coupling of ligand binding to channel opening. Here, we investigated an allosteric binding site in the ion channel vestibule, which has converged from a series of studies on prokaryote and eukaryote channel homologs. We discovered single domain antibodies, called nanobodies, which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote channel ELIC bound either to a positive (PAM) or a negative (NAM) allosteric modulator. We extrapolate the functional importance of the vestibule binding site to eukaryote ion channels, suggesting a conserved mechanism of allosteric modulation. This work identifies key elements of allosteric binding sites and extends drug design possibilities in pLGICs using nanobodies.

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In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Molecules ◽

10.3390/molecules27020453 ◽

2022 ◽

Vol 27 (2) ◽

pp. 453

Author(s):

Jiayi Yuan ◽

Chen Jiang ◽

Junmei Wang ◽

Chih-Jung Chen ◽

Yixuan Hao ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Cannabinoid Receptors ◽

Md Simulations ◽

Docking Studies ◽

Allosteric Modulators ◽

3D Structures ◽

Potential Binding ◽

Allosteric Sites ◽

Allosteric Binding Sites

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

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Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418289112 ◽

2015 ◽

Vol 112 (19) ◽

pp. E2543-E2552 ◽

Cited By ~ 65

Author(s):

Radovan Spurny ◽

Sarah Debaveye ◽

Ana Farinha ◽

Ken Veys ◽

Ann M. Vos ◽

...

Keyword(s):

Binding Site ◽

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Binding Sites ◽

Agonist Binding ◽

Α7 Nicotinic Acetylcholine Receptor ◽

Nicotinic Acetylcholine ◽

Α7 Nachr ◽

Allosteric Binding Sites ◽

Α Helix

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

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Loop C and the mechanism of acetylcholine receptor–channel gating

The Journal of General Physiology ◽

10.1085/jgp.201210946 ◽

2013 ◽

Vol 141 (4) ◽

pp. 467-478 ◽

Cited By ~ 52

Author(s):

Prasad Purohit ◽

Anthony Auerbach

Keyword(s):

Binding Site ◽

Binding Sites ◽

Single Channel ◽

Communication Link ◽

Channel Opening ◽

State Models ◽

Open Question ◽

Acetylcholine Receptor Channel ◽

Global Transition ◽

Receptor Channel

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.

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Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

10.26434/chemrxiv.9853445 ◽

2019 ◽

Author(s):

Patrick R. A. Zanon ◽

Lisa Lewald ◽

Stephan M. Hacker

Keyword(s):

Binding Sites ◽

Bacterial Resistance ◽

Mevalonate Pathway ◽

Rapid Development ◽

High Reactivity ◽

Functional Importance ◽

Essential Proteins ◽

Covalent Inhibitors ◽

Druggable Targets ◽

Covalent Ligands

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in Staphylococcus aureus and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-action.

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Covalent-Fragment Screening of Brd4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites

10.26434/chemrxiv.8859098 ◽

2019 ◽

Author(s):

Michael Olp ◽

Daniel Sprague ◽

Stefan Kathman ◽

Ziyang Xu ◽

Alexandar Statsyuk ◽

...

Keyword(s):

Mass Spectrometry ◽

Binding Site ◽

Binding Sites ◽

Computational Prediction ◽

Chemical Probes ◽

Computational Docking ◽

Fragment Screening ◽

Covalent Inhibitors ◽

Bet Protein ◽

Proof Of Principle

Brd4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as a promising epigenetic target in cancer and inflammatory disorders. All reported BET family ligands bind within the bromodomain acetyl-lysine binding sites and competitively inhibit BET protein interaction with acetylated chromatin. Alternative chemical probes that act orthogonally to the highly-conserved acetyl-lysine binding sites may exhibit selectivity within the BET family and avoid recently reported toxicity in clinical trials of BET bromodomain inhibitors. Here, we report the first identification of a ligandable site on a bromodomain outside the acetyl-lysine binding site. Inspired by our computational prediction of hotspots adjacent to non-homologous cysteine residues within the C-terminal Brd4 bromodomain (Brd4-BD2), we performed a mid-throughput mass spectrometry screen to identify cysteine-reactive fragments that covalently and selectively modify Brd4. Subsequent mass spectrometry, NMR and computational docking analyses of electrophilic fragment hits revealed a novel ligandable site near Cys356 that is unique to Brd4 among all human bromodomains. This site is orthogonal to the Brd4-BD2 acetyl-lysine binding site as Cys356 modification did not impact binding of the pan-BET bromodomain inhibitor JQ1 in fluorescence polarization assays. Finally, we tethered covalent fragments to JQ1 and performed NanoBRET assays to provide proof of principle that this orthogonal site can be covalently targeted in intact human cells. Overall, we demonstrate the potential of targeting sites orthogonal to bromodomain acetyl-lysine binding sites to develop bivalent and covalent inhibitors that displace Brd4 from chromatin.

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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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Tuning of Electronic Properties in Conducting Polymers

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20011208 ◽

2001 ◽

Vol 66 (8) ◽

pp. 1208-1218 ◽

Cited By ~ 3

Author(s):

Guofeng Li ◽

Mira Josowicz ◽

Jiří Janata

Keyword(s):

Hydrogen Bonding ◽

Binding Site ◽

Binding Sites ◽

Polymer Chains ◽

Electronic Transitions ◽

Weakly Bound ◽

Ordered Structures ◽

Doped Polymer ◽

Positively Charged ◽

Repeated Cycling

Structural and electronic transitions in poly(thiophenyleneiminophenylene), usually referred to as poly(phenylenesulfidephenyleneamine) (PPSA) upon electrochemical doping with LiClO4 have been investigated. The unusual electrochemical behavior of PPSA indicates that the dopant anions are bound in two energetically different sites. In the so-called "binding site", the ClO4- anion is Coulombically attracted to the positively charged S or N sites on one chain and simultaneously hydrogen-bonded with the N-H group on a neighboring polymer chain. This strong interaction causes a re-organization of the polymer chains, resulting in the formation of a networked structure linked together by these ClO4- Coulombic/hydrogen bonding "bridges". However, in the "non-binding site", the ClO4- anion is very weakly bound, involves only the electrostatic interaction and can be reversibly exchanged when the doped polymer is reduced. In the repeated cycling, the continuous and alternating influx and expulsion of ClO4- ions serves as a self-organizing process for such networked structures, giving rise to a diminishing number of available "non-binding" sites. The occurrence of these ordered structures has a major impact on the electrochemical activity and the morphology of the doped polymer. Also due to stabilization of the dopant ions, the doped polymer can be kept in a stable and desirable oxidation state, thus both work function and conductivity of the polymer can be electrochemically controlled.

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Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5805 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5805-5813 ◽

Cited By ~ 52

Author(s):

M M Wang ◽

R Y Tsai ◽

K A Schrader ◽

R R Reed

Keyword(s):

Signal Transduction ◽

Dna Binding ◽

Binding Site ◽

Binding Sites ◽

Consensus Sequence ◽

Initiation Site ◽

Olfactory Neuron ◽

Promoter Regions ◽

Transcriptional Initiation ◽

Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

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