| 1. |
- Haloi, Nandan, et al.
(författare)
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Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids
- 2024
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 37
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Tidskriftsartikel (refereegranskat)abstract
- We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for ∆F/F0 vs [nicotine] (δ-slope, 2.7 μM−1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the ‘candle snuffer’ mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.
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| 2. |
- Bergh, Cathrine, et al.
(författare)
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Discovery of lipid binding sites in a ligand-gated ion channel by integrating simulations and cryo-EM
- 2024
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Ingår i: eLife. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 12, s. 2023-01
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Tidskriftsartikel (refereegranskat)abstract
- Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside fromcanonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ionchannels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution ofthese regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLICby integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipidsin the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipidswere associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutantsimulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, whilea putative intersubunit site was preferred in open-state simulations. This work offers molecular details ofGLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependentbinding, and a multidisciplinary strategy for modeling protein-lipid interactions.
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| 3. |
- Cowgill, John, et al.
(författare)
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Structure and dynamics of differential ligand binding in the human ρ-type GABAA receptor
- 2023
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Ingår i: Neuron. - : Elsevier BV. - 0896-6273 .- 1097-4199. ; 111:21, s. 5-3450
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Tidskriftsartikel (refereegranskat)abstract
- The neurotransmitter γ-aminobutyric acid (GABA) drives critical inhibitory processes in and beyond the nervous system, partly via ionotropic type-A receptors (GABAARs). Pharmacological properties of ρ-type GABAARs are particularly distinctive, yet the structural basis for their specialization remains unclear. Here, we present cryo-EM structures of a lipid-embedded human ρ1 GABAAR, including a partial intracellular domain, under apo, inhibited, and desensitized conditions. An apparent resting state, determined first in the absence of modulators, was recapitulated with the specific inhibitor (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid and blocker picrotoxin and provided a rationale for bicuculline insensitivity. Comparative structures, mutant recordings, and molecular simulations with and without GABA further explained the sensitized but slower activation of ρ1 relative to canonical subtypes. Combining GABA with picrotoxin also captured an apparent uncoupled intermediate state. This work reveals structural mechanisms of gating and modulation with applications to ρ-specific pharmaceutical design and to our biophysical understanding of ligand-gated ion channels.
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| 4. |
- Gao, Xiaolong, et al.
(författare)
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Allosteric inhibition of CFTR gating by CFTRinh-172 binding in the pore
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR’s activity through binding to a site also comprising TM8.
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| 5. |
- Haug, Ferdinand M., et al.
(författare)
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Functional and structural insights into activation of TRPV2 by weak acids
- 2024
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Ingår i: EMBO Journal. - 0261-4189 .- 1460-2075. ; 43:11, s. 2264-2290
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Tidskriftsartikel (refereegranskat)abstract
- Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.
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| 6. |
- Jalalypour, Farzaneh, et al.
(författare)
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Allosteric Cholesterol Site in Glycine Receptors Characterized through Molecular Simulations
- 2024
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 128:20, s. 4996-5007
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Tidskriftsartikel (refereegranskat)abstract
- Glycine receptors are pentameric ligand-gated ion channels that conduct chloride ions across postsynaptic membranes to facilitate fast inhibitory neurotransmission. In addition to gating by the glycine agonist, interactions with lipids and other compounds in the surrounding membrane environment modulate their function, but molecular details of these interactions remain unclear, in particular, for cholesterol. Here, we report coarse-grained simulations in a model neuronal membrane for three zebrafish glycine receptor structures representing apparent resting, open, and desensitized states. We then converted the systems to all-atom models to examine detailed lipid interactions. Cholesterol bound to the receptor at an outer-leaflet intersubunit site, with a preference for the open and desensitized versus resting states, indicating that it can bias receptor function. Finally, we used short atomistic simulations and iterative amino acid perturbations to identify residues that may mediate allosteric gating transitions. Frequent cholesterol contacts in atomistic simulations clustered with residues identified by perturbation analysis and overlapped with mutations influencing channel function and pathology. Cholesterol binding at this site was also observed in a recently reported pig heteromeric glycine receptor. These results indicate state-dependent lipid interactions relevant to allosteric transitions of glycine receptors, including specific amino acid contacts applicable to biophysical modeling and pharmaceutical design.
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| 7. |
- Krahn, Natalie, et al.
(författare)
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tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 52:2, s. 513-524
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Tidskriftsartikel (refereegranskat)abstract
- Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating >200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G-U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G-C base pairs (G3:C70 and G5:C68).
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| 8. |
- Legesse, Dagimhiwat H., et al.
(författare)
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Structural insights into opposing actions of neurosteroids on GABAA receptors
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- γ-Aminobutyric acid type A (GABAA) receptors mediate fast inhibitory signaling in the brain and are targets of numerous drugs and endogenous neurosteroids. A subset of neurosteroids are GABAA receptor positive allosteric modulators; one of these, allopregnanolone, is the only drug approved specifically for treating postpartum depression. There is a consensus emerging from structural, physiological and photolabeling studies as to where positive modulators bind, but how they potentiate GABA activation remains unclear. Other neurosteroids are negative modulators of GABAA receptors, but their binding sites remain debated. Here we present structures of a synaptic GABAA receptor bound to allopregnanolone and two inhibitory sulfated neurosteroids. Allopregnanolone binds at the receptor-bilayer interface, in the consensus potentiator site. In contrast, inhibitory neurosteroids bind in the pore. MD simulations and electrophysiology support a mechanism by which allopregnanolone potentiates channel activity and suggest the dominant mechanism for sulfated neurosteroid inhibition is through pore block.
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| 9. |
- Zhuang, Yuxuan, et al.
(författare)
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Symmetry-Adapted Markov State Models of Closing, Opening, and Desensitizing in α7 nicotinic acetylcholine receptors
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The α7 nicotinic acetylcholine receptors (nAChRs) are homopentameric ligand-gated ion channels gated by the neurotransmitter acetylcholine. These receptors play a crucial role in controlling electrical signaling within the nervous system by facilitating the passage of cations across the membrane. Recent studies have resolved and functionally annotated closed, open, and desensitized states of α7 nAChRs, providing insight into ion permeation and lipid modulation effects. However, the process by which α7 nAChRs transition between states remains unclear. To better understand gating and lipid modulation, we generated two ensembles of molecular dynamics simulations of the apo form of α7 nAChRs, with or without cholesterol. Using symmetry-adapted Markov state modeling, we developed a five-state gating model. As expected for the unliganded condition, the channel predominantly resides in its closed state. The kinetics of the transition from open to one non-conductive intermediate (flipped) state corresponded to an experimentally-measured opening duration of 0.1 ms. The addition of cholesterol led to the stabilization of the desensitized state, and strengthened the coupling between various states. These results establish plausible asymmetric transition pathways between functionally important states, and define lipid modulation effects in the conformational cycle in α7 nAChRs, as well as providing an ensemble of structural models that could be utilized for future rational lipidic drug design.
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