Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades

Delgado-Vélez, Manuel; Quesada, Orestes; Villalobos‐Santos, Juan C.; Maldonado‐Hernández, Rafael; Asmar-Rovira, Guillermo; Stevens, Raymond C.; Lasalde‐Dominicci, José A.

doi:10.3390/molecules26195753

Cited by 12 publications

(11 citation statements)

References 174 publications

(222 reference statements)

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“…N-linked glycosylation can significantly change the conformation of proteins and subsequently influence the interaction and function of proteins ( Delgado-Vélez et al, 2021 ). Therefore, the analysis of N-glycoproteins interaction is important for the functional study of these N-glycoproteins.…”

Section: Resultsmentioning

confidence: 99%

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

Xie

Chen

et al. 2021

Front. Plant Sci.

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N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

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Section: Resultsmentioning

confidence: 99%

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

Xie

Chen

et al. 2021

Front. Plant Sci.

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“…Overall, these results indicate that the nAChR can be stable and functional in lipid-analog detergents or in detergents that retain moderate amounts of residual native lipids, while the opposite is true about non-lipid-analog detergents. These data highlight the importance of a careful biophysical characterization of the membrane protein-detergent complex (MP-DCs) for future structural studies (Hamouda et al 2006a , b ; Maldonado-Hernández et al 2020 ; Delgado-Vélez et al 2021 ).…”

Section: Discussionmentioning

confidence: 97%

“…The first obstacle in achieving high-resolution, X-ray structures of the nAChRs is the preparation of milligram amounts of pure, homogeneous, functional, and stable nAChR-detergent complexes (nAChR-DCs). Other difficulties in crystalizing nAChRs are: (1) heterogenic pentamers, (2) multiple stoichiometries, (3) pseudosimetry of heteropentamers, (4) glycosylation of extracellular domains with diverse sugar compositions, (5) large intracellular domains (M3-M4 loop) with disordered structure, and (6) different conformations (Asmar-Rovira et al 2008 ; Delgado-Vélez et al 2021 ). Along these lines, the preparation and the reproducibility of nAChRs protein crystals suitable for X-ray diffraction studies have become remarkably challenging experimentally and the foremost obstacle to attaining high-resolution structures.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

et al. 2023

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The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies. Graphical abstract

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“…In recent years, there has been a breakthrough in the use of cryo-electron microscopy for nAChRs, which allowed resolution of the structures of the muscle-type receptor from the Torpedo ray electric organ [ 8 ], the neuronal human α4β2 nAChR in complex with nicotine and antibody fragments specific for the β2-subunit [ 179 ], the α3β4 receptor also in complex with agonists but already in a lipid environment [ 180 ], as well as α7 nAChR in three different states (apo, agonist-bound and agonist/PNU-120596 bound) [ 181 ]. It is important that over the past two years, cryo-electron microscopy structures of nicotinic receptors have also been obtained in complex with the polypeptide antagonist α-bungarotoxin from the banded krait venom, namely the muscle-type receptor from the Torpedo electric organ [ 9 ] and the neuronal human α7 nAChR [ 10 ] (for more detailed high resolution structural studies of full-size nicotinic receptors, see, for example, the review [ 182 ]). Probably, the respective structures with peptide ligands should be expected in the near future.…”

Section: Marine Origin Peptides Targeting Nachrsmentioning

confidence: 99%

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

et al. 2022

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The purpose of our review is to briefly show what different compounds of marine origin, from low molecular weight ones to peptides and proteins, offer for understanding the structure and mechanism of action of nicotinic acetylcholine receptors (nAChRs) and for finding novel drugs to combat the diseases where nAChRs may be involved. The importance of the mentioned classes of ligands has changed with time; a protein from the marine snake venom was the first excellent tool to characterize the muscle-type nAChRs from the electric ray, while at present, muscle and α7 receptors are labeled with the radioactive or fluorescent derivatives prepared from α-bungarotoxin isolated from the many-banded krait. The most sophisticated instruments to distinguish muscle from neuronal nAChRs, and especially distinct subtypes within the latter, are α-conotoxins. Such information is crucial for fundamental studies on the nAChR revealing the properties of their orthosteric and allosteric binding sites and mechanisms of the channel opening and closure. Similar data are provided by low-molecular weight compounds of marine origin, but here the main purpose is drug design. In our review we tried to show what has been obtained in the last decade when the listed classes of compounds were used in the nAChR research, applying computer modeling, synthetic analogues and receptor mutants, X-ray and electron-microscopy analyses of complexes with the nAChRs, and their models which are acetylcholine-binding proteins and heterologously-expressed ligand-binding domains.

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Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades

Cited by 12 publications

References 174 publications

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

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