<i>In situ</i> ligand restraints from quantum-mechanical methods

Liebschner, Dorothée; Moriarty, Nigel W.; Poon, Billy K.; Adams, Paul D.

doi:10.1107/s2059798323000025

Cited by 10 publications

(6 citation statements)

References 45 publications

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“…The models were further refined with ISOLDE (Croll, 2018) in ChimeraX (Pettersen et al, 2004) (v.1.6) and Phenix (Liebschner et al, 2019) (v1.20) real space refinement. The two potential conformations for the AK-42 ligand were tested using the Quantum Mechanical Restraints (QMR) method (Liebschner et al, 2023). Q-scores were performed with the MapQ Chimera plugin v1.9.12.…”

Section: Methodsmentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a ball-and-chain gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

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Section: Methodsmentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Two conformations were found to fit the density well with the carboxyl group on opposite sides. They were tested using the Quantum Mechanical Restraints (QMR) method (Liebschner et al, 2023 ); one had significantly lower energy (∼17 kcal/mol) and strain on the ligand (10.2 kcal/mol), considering the ligand and nearby residues only. For the lower energy conformation, the Q-scores of AK-42 in the map are 0.74 and 0.76 in the two subunits, respectively.…”

Section: Ak-42 Bound Structurementioning

confidence: 99%

“…The models were further refined with ISOLDE (Croll, 2018 ) in ChimeraX (Pettersen et al, 2004 ) (v.1.6) and Phenix (Liebschner et al, 2019 ) (v1.20) real space refinement. The two potential conformations for the AK-42 ligand were tested using the Quantum Mechanical Restraints (QMR) method (Liebschner et al, 2023 ). Q-scores were performed with the MapQ Chimera plugin v1.9.12.…”

Section: Model Building and Refinementmentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

Xu,

Neelands,

Powers

et al. 2024

eLife

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl−-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl−-permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl−-permeation pathway.

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“…In addition, many researchers generate their own restraints using the computational method available to them. Generating correct restrains for metal coordinating molecules (which are common enzyme cofactors) is difficult and constitutes its own, yet unresolved, challenge. , In addition, and in contrast to the 20 natural amino acids, high quality ligand validation in deposited structures is problematic and still under development.…”

Section: Introductionmentioning

confidence: 99%

“…Generating correct restrains for metal coordinating molecules (which are common enzyme cofactors) is difficult and constitutes its own, yet unresolved, challenge. 26,27 In addition, and in contrast to the 20 natural amino acids, high quality ligand validation in deposited structures is problematic and still under development. A notable success in characterizing the functional significance of ligand deformation comes from the heme group found in oxygen-transport proteins such as hemoglobin and myoglobin and in redox-active proteins such as cytochromes.…”

Section: ■ Introductionmentioning

confidence: 99%

Feeling the Strain: Quantifying Ligand Deformation in Photosynthesis

Lin,

Mazor,

Reppert

2024

J. Phys. Chem. B

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Structural distortion of protein-bound ligands can play a critical role in enzyme function by tuning the electronic and chemical properties of the ligand molecule. However, quantifying these effects is difficult due to the limited resolution of protein structures and the difficulty of generating accurate structural restraints for nonprotein ligands. Here, we seek to quantify these effects through a statistical analysis of ligand distortion in chlorophyll proteins (CP), where ring deformation is thought to play a role in energy and electron transfer. To assess the accuracy of ring-deformation estimates from available structural data, we take advantage of the C 2 symmetry of photosystem II (PSII), comparing ring-deformation estimates for equivalent sites both within and between 113 distinct X-ray and cryogenic electron microscopy PSII structures. Significantly, we find that several deformation modes exhibit considerable variability in predictions, even for equivalent monomers, down to a 2 Å resolution, to an extent that probably prevents their utilization in optical calculations. We further find that refinement restraints play a critical role in determining deformation values to resolution as low as 2 Å. However, for those modes that are well-resolved in the structural data, ring deformation in PSII is strongly conserved across all species tested from cyanobacteria to algae. These results highlight both the opportunities and limitations inherent in structure-based analyses of the bioenergetic and optical properties of CPs and other protein−ligand complexes.

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In situ ligand restraints from quantum-mechanical methods

Cited by 10 publications

References 45 publications

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

Feeling the Strain: Quantifying Ligand Deformation in Photosynthesis

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