scholarly journals Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel

2016 ◽  
Vol 113 (5) ◽  
pp. E644-E653 ◽  
Author(s):  
Nurit Degani-Katzav ◽  
Revital Gortler ◽  
Lilach Gorodetzki ◽  
Yoav Paas
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Parasitic Diseases ◽  
Β Subunit ◽  
River Blindness ◽  
Subunit Stoichiometry ◽  
Binding Pockets ◽  
Extracellular Side ◽  
Electrophysiological Characterization

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the “principal” and “complementary” components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits’ cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

scholarly journals Hybrid Dynamic Pharmacophore Models as Effective Tools to Identify Novel Chemotypes for Anti-TB Inhibitor Design: A Case Study With Mtb-DapB

2020 ◽  
Vol 8 ◽  
Author(s):  
Chinmayee Choudhury ◽  
Anshu Bhardwaj
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Chemical Space ◽  
Pharmacophore Model ◽  
Model Systems ◽  
Receptor Interaction ◽  
Hybrid Molecules ◽  
Wide Range ◽  
Binding Pockets ◽  
Pharmacophore Models

Antimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200 ns molecular dynamics simulations and trajectories were analyzed to identify stable ligand-receptor interaction features. Based on these interactions, conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 1,563,764 publicly available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4-point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

scholarly journals Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kangcheng Song ◽  
Miao Wei ◽  
Wenjun Guo ◽  
Li Quan ◽  
Yunlu Kang ◽  
...  
Keyword(s):  
Anxiety Disorder ◽  
Binding Sites ◽  
Structural Basis ◽  
Inhibitory Mechanism ◽  
Inhibitor Binding ◽  
Design And Optimization ◽  
Physiological Processes ◽  
Channel Inhibition ◽  
Binding Pockets ◽  
Extracellular Side

TRPC5 channel is a non-selective cation channel that participates diverse physiological processes. TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression and kidney disease. However, the binding sites and inhibitory mechanism of TRPC5 inhibitors remain elusive. Here we present the cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 Å. The structures reveal that clemizole binds inside the voltage sensor-like domain of each subunit. In contrast, HC-070 is wedged between adjacent subunits and replaces the glycerol group of a putative DAG molecule near the extracellular side. Moreover, we found mutations in the inhibitor binding pockets altered the potency of inhibitors. These structures suggest that both clemizole and HC-070 exert the inhibitory functions by stabilizing the ion channel in a non-conductive closed state. These results pave the way for further design and optimization of inhibitors targeting human TRPC5.

scholarly journals MK-801 is a potent nematocidal agent. Characterization of MK-801 binding sites in Caenorhabditis elegans

1989 ◽  
Vol 260 (3) ◽  
pp. 923-926 ◽  
Author(s):  
J M Schaeffer ◽  
A R Bergstrom ◽  
M J Turner
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Site ◽  
Binding Sites ◽  
Mammalian Brain ◽  
Apparent Dissociation Constant ◽  
Mk 801 ◽  
C Elegans ◽  
Mammalian Tissues ◽  
Free Living Nematode

MK-801, an N-methyl-D-aspartate antagonist in mammalian brain tissue, is a potent nematocidal agent. Specific MK-801 binding sites have been identified and characterized in a membrane fraction prepared from the free-living nematode Caenorhabditis elegans. The high-affinity MK-801 binding site has an apparent dissociation constant, Kd, of 225 nM. Unlike the MK-801 binding site in mammalian tissues, the C. elegans binding site is not effected by glutamate or glycine, and polyamines are potent inhibitors of specific MK-801 binding.

scholarly journals Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene.

1988 ◽  
Vol 8 (8) ◽  
pp. 3215-3226 ◽  
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
S S Banerji ◽  
M Sheffery
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Globin Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Murine Erythroleukemia

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

scholarly journals A single historical substitution drives an increase in acetylcholine receptor complexity

2021 ◽  
Vol 118 (7) ◽  
pp. e2018731118
Author(s):  
Johnathon R. Emlaw ◽  
Christian J. G. Tessier ◽  
Gregory D. McCluskey ◽  
Melissa S. McNulty ◽  
Yusuf Sheikh ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acetylcholine Receptor ◽  
Amino Acid Substitution ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Single Amino Acid ◽  
Β Subunit ◽  
Subunit Stoichiometry ◽  
Sequence Reconstruction

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral β-subunit can not only replace the extant β-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral β-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.

scholarly journals Hybrid dynamic pharmacophore models as effective tools to identify novel chemotypes for anti-TB inhibitor design: A case study with Mtb-DapB

2020 ◽  
Author(s):  
Chinmayee Choudhury ◽  
Anshu Bhardwaj
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Chemical Space ◽  
Pharmacophore Model ◽  
Model Systems ◽  
Receptor Interaction ◽  
Hybrid Molecules ◽  
Wide Range ◽  
Binding Pockets ◽  
Pharmacophore Models

AbstractAntimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200ns molecular dynamics simulations and trajectories were analyzed to identify stable ligand-receptor interaction features. Based on these interactions, conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 15, 63,764 publicly available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4-point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene

1988 ◽  
Vol 8 (8) ◽  
pp. 3215-3226
Author(s):  
K M Barnhart ◽  
C G Kim ◽  
S S Banerji ◽  
M Sheffery
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Binding Sites ◽  
Globin Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Erythroid Cell ◽  
Alpha Globin ◽  
Murine Erythroleukemia

The proteins responsible for erythroid-specific footprints extending to -180 on the mouse alpha-globin gene were identified, enriched, and characterized from extracts of murine erythroleukemia (MEL) cells. Three proteins accounted for most aspects of the footprints. The binding sites of two proteins, termed alpha-CP1 and alpha-CP2, overlapped in the CCAAT box. Further characterization of these two CCAAT binding proteins showed that neither interacted with the adenovirus origin of replication, a strong CCAAT transcription factor-nuclear factor 1 binding site. A third protein, termed alpha-IRP, interacted with two sequences that formed an inverted repeat (IR) between the CCAAT and TATAA boxes. Interestingly, the binding domain of one of the CCAAT factors, alpha-CP1, overlapped one alpha-IRP binding site. alpha-CP1 thus overlapped the binding domains of both alpha-CP2 and alpha-IRP. The IRs included GC-rich sequences reminiscent of SP1-binding sites. Indeed, alpha-IRP bound as well to the alpha-promoter as it did to SP1 sites in the simian virus 40 early promoter. These results suggest that alpha-IRP may be related to the transcription factor Sp1. We determined the level of each alpha-globin-binding activity before and after induced erythroid differentiation of MEL cells. We found that differentiation caused alpha-CP1 activity to drop three- to fivefold, while alpha-IRP activity decreased slightly and alpha-CP2 activity increased two- to threefold.

scholarly journals Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families.

1981 ◽  
Vol 154 (5) ◽  
pp. 1584-1598 ◽  
Author(s):  
C M Andres ◽  
A Maddalena ◽  
S Hudak ◽  
N M Young ◽  
J L Claflin
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Structural Heterogeneity ◽  
The Other ◽  
Family Diversity ◽  
Differential Reactivity ◽  
Myeloma Proteins ◽  
Rational Explanation ◽  
Intra Family

The present investigation extends our immunochemical characterization of binding site heterogeneity among a large series of monoclonal anti-phosphocholine (PC) antibodies. Hybridoma proteins (HP) from eight genetically distinct strains are included in this study, yet no strain specific characteristics are observed. These HP, as previously shown (5), are divided into three well-defined families based on public or family-specific Id and L chain isotypes characteristic of three PC-binding myeloma proteins: T15, M603, and M511. All antibodies exhibited some degree of inter- or intra-family heterogeneity, or both. Some of this intra-family diversity was reflected by differential reactivity for PC when attached to three different carriers. In spite of this, the specificity profiles for hapten analogues of PC, as measured by hapten inhibition of binding, were the same for all members of the T15 family. Altering the carrier had no effect, thus suggesting that the binding site pocket for PC is essentially preserved, whereas that for carrier is variable. Similar conclusions were reached for most of the M603 HP, although the binding site is different from the T15 HP. The M511 HP stand in sharp contrast to the HP in the other two families because their binding sites exhibit extensive variability. The independence in reactivity for PC and PC plus carrier offers a rational explanation for idiotypic and/or structural heterogeneity within a family. More importantly it suggests interesting strategies for diversification within one group of antibodies.

scholarly journals Hybrid Dynamic Pharmacophore Models as Effective Tools to Identify Novel Chemotypes for Anti-tb Inhibitor Design: a Case Study With Mtb-dapb

2020 ◽  
Author(s):  
Chinmayee Choudhury ◽  
Anshu Bhardwaj
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Chemical Space ◽  
Pharmacophore Model ◽  
Model Systems ◽  
Receptor Interaction ◽  
Hybrid Molecules ◽  
Wide Range ◽  
Binding Pockets ◽  
Pharmacophore Models

Abstract Antimicrobial resistance (AMR) is one of the most serious global public health threats as it compromises the successful treatment of deadly infectious diseases like tuberculosis. New therapeutics are constantly needed but it takes a long time and is expensive to explore new biochemical space. One way to address this issue is to repurpose the validated targets and identify novel chemotypes that can simultaneously bind to multiple binding pockets of these targets as a new lead generation strategy. This study reports such a strategy, dynamic hybrid pharmacophore model (DHPM), which represents the combined interaction features of different binding pockets contrary to the conventional approaches, where pharmacophore models are generated from single binding sites. We have considered Mtb-DapB, a validated mycobacterial drug target, as our model system to explore the effectiveness of DHPMs to screen novel unexplored compounds. Mtb-DapB has a cofactor binding site (CBS) and an adjacent substrate binding site (SBS). Four different model systems of Mtb-DapB were designed where, either NADPH/NADH occupies CBS in presence/absence of an inhibitor 2, 6-PDC in the adjacent SBS. Two more model systems were designed, where 2, 6-PDC was linked to NADPH and NADH to form hybrid molecules. The six model systems were subjected to 200ns molecular dynamics simulations and trajectories were analysed to identify stable ligand-receptor interaction features. Based on these interactions, Conventional pharmacophore models (CPM) were generated from the individual binding sites while DHPMs were created from hybrid-molecules occupying both binding sites. A huge library of 15, 63,764 publically available molecules were screened by CPMs and DHPMs. The screened hits obtained from both types of models were compared based on their Hashed binary molecular fingerprints and 4 point pharmacophore fingerprints using Tanimoto, Cosine, Dice and Tversky similarity matrices. Molecules screened by DHPM exhibited significant structural diversity, better binding strength and drug like properties as compared to the compounds screened by CPMs and the reported anti-mycobacterial molecules indicating the efficiency of DHPM to explore new chemical space for anti-TB drug discovery. The idea of DHPM can be applied for a wide range of mycobacterial or other pathogen targets to venture into unexplored chemical space.

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