Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism
<p>Pharmacological actions of a molecular switch moiety are reproduced in sub-microsecond MD simulations in GIRK1/2. (<b>A</b>) The structures of GIRK1 specific modulators ML297, GAT1587, and the site of molecular switching region (off the pyrazole ring) that controls compound activity. (<b>B</b>) Equilibrium binding site for ML297 and GAT1587 following a 300 ns stochastic dynamics simulation in complex with PIP<sub>2</sub> and GIRK1/2. (<b>C</b>) Ion permeation pathway for a single ion taken from the ML297-PIP2-GIRK1/2 simulation. (<b>D</b>–<b>F</b>) Ions conducted, minimum gate distances, and normalized salt-bridge formation over the last 150 ns of the simulations are shown.</p> "> Figure 2
<p>GAT1587 binding site along the TM helices in the GIRK1/2 heterotetramer.(<b>A</b>) Equilibrium binding pose for ML297 when complexed with PIP<sub>2</sub> GIRK1/2 after 300 ns of stochastic dynamics. (<b>B</b>) A 2D schematic representation of the compound’s binding pose depicting protein ligand interactions. (<b>C</b>) Equilibrium binding pose for GAT1587 when complexed with PIP<sub>2</sub> GIRK1/2 after 300 ns of stochastic dynamics. (<b>D</b>) A 2D schematic representation of the compounds binding pose depicting protein ligand interactions.</p> "> Figure 3
<p>ML297 decreases and GAT1587 increases the angle between the outer and inner TM2 segments on each side of the flexible GIRK1-G169. (<b>A</b>) Comparison between TM2 of the ML297 (dark orange) and the GAT1587 (light orange) complexed with the GIRK1/2 systems. (<b>B</b>) The two hinge points that allow for TM2 bending. (<b>C</b>) Ramachandran plots for residues within TM2. I167 is highlighted in both. (<b>D</b>) Schematic representation of the movements of TM2 and a quantification of the bending around GIRK1-G169. (<b>E</b>) Expanded model including the nearby TM1 and a quantification of these effects. (<b>F</b>) Relative movements of the two transmembrane helices.</p> "> Figure 4
<p>A hydrophobic wire (W95-Y91-F87) couples TM1 to TM2 at the level of the HBC gate (M180) and is stabilized by ML297 but not by GAT1587 to regulate the E141/D173-dependent conduction. (<b>A</b>) Protein structure with the pi-stack along TM1 highlighted. (<b>B</b>) Interaction distributions in the presence of ML297 or GAT1587. Pink denotes pi–pi interactions with an upper cutoff of 4 angstroms. Light pink denotes Van der Waals interactions with an upper cutoff of 3 angstroms. (<b>C</b>) Protein structure with the stabilizing hydrophobic residues of the GIRK2 subunit that can interact with the TM1 hydrophobic wire residues. (<b>D</b>) Interaction distance distributions of hydrophobic residues involved when either ML297 or GAT1587 is present. Light pink denotes Van der Waals interactions with an upper cutoff of 3 angstroms. (<b>E</b>) Protein structure (<b>E1</b>) with acidic residues that interact with a potassium ion (highlighted interactions) (<b>E2</b>). Dotted lines indicate a broken interaction between residues. The solid lines indicate the formation of an interaction. (<b>F</b>) Interaction distance distributions of TM1 hydrophobic chain residues with the two residues of the dipole between D141 and D173 of GIRK1 and S181 (next to G180) of GIRK2 in the presence of ML297 or GAT1587. Light green denotes dipole integrations with an upper cutoff of 4 angstroms. (<b>G</b>) Schematic representation of the charge relay network between W95 and Y91 of the TM1 hydrophobic chain with the two acidic residues.</p> "> Figure 4 Cont.
<p>A hydrophobic wire (W95-Y91-F87) couples TM1 to TM2 at the level of the HBC gate (M180) and is stabilized by ML297 but not by GAT1587 to regulate the E141/D173-dependent conduction. (<b>A</b>) Protein structure with the pi-stack along TM1 highlighted. (<b>B</b>) Interaction distributions in the presence of ML297 or GAT1587. Pink denotes pi–pi interactions with an upper cutoff of 4 angstroms. Light pink denotes Van der Waals interactions with an upper cutoff of 3 angstroms. (<b>C</b>) Protein structure with the stabilizing hydrophobic residues of the GIRK2 subunit that can interact with the TM1 hydrophobic wire residues. (<b>D</b>) Interaction distance distributions of hydrophobic residues involved when either ML297 or GAT1587 is present. Light pink denotes Van der Waals interactions with an upper cutoff of 3 angstroms. (<b>E</b>) Protein structure (<b>E1</b>) with acidic residues that interact with a potassium ion (highlighted interactions) (<b>E2</b>). Dotted lines indicate a broken interaction between residues. The solid lines indicate the formation of an interaction. (<b>F</b>) Interaction distance distributions of TM1 hydrophobic chain residues with the two residues of the dipole between D141 and D173 of GIRK1 and S181 (next to G180) of GIRK2 in the presence of ML297 or GAT1587. Light green denotes dipole integrations with an upper cutoff of 4 angstroms. (<b>G</b>) Schematic representation of the charge relay network between W95 and Y91 of the TM1 hydrophobic chain with the two acidic residues.</p> "> Figure 5
<p>The ML297-induced SH movement, as a result of the TM1 movement, drives changes in the CD loop interactions causing stabilization of the G-loop in the open conformation through its residue GIRK2-E315.2 liberating K188.1 that coordinates PIP<sub>2</sub> to stabilize the HBC gate in the open conformation. (<b>A</b>) A model of how TM1 modulates the slide helix (<b>A1</b>) and a quantification of these effects (<b>A2</b>). (<b>B</b>) Relative movements of the activated and inhibited SH regions. (<b>C</b>) Protein structure (<b>C1</b>) with residues that link the SH to the CD loop (<b>C2</b>). (<b>D</b>) Distance distributions of key residues that drive the channel into the active state. Dark yellow denotes charge–charge interactions with an upper cutoff of 4.0 angstroms. Light green denotes dipole charge interactions with an upper cut off of 4.0 angstroms. (<b>E</b>) Schematic outline of the changes in key residue interactions. (<b>F</b>) Protein structure (<b>F1</b>) with residues that link the SH to the CD loop (<b>F2</b>). (<b>G</b>) Distance distributions of residues that drive the channel into the active state. Dark yellow denotes charge–charge interactions with an upper cutoff of 4.0 angstroms. Pink denotes dipole charge interactions with an upper cutoff of 4.0 angstroms.</p> "> Figure 5 Cont.
<p>The ML297-induced SH movement, as a result of the TM1 movement, drives changes in the CD loop interactions causing stabilization of the G-loop in the open conformation through its residue GIRK2-E315.2 liberating K188.1 that coordinates PIP<sub>2</sub> to stabilize the HBC gate in the open conformation. (<b>A</b>) A model of how TM1 modulates the slide helix (<b>A1</b>) and a quantification of these effects (<b>A2</b>). (<b>B</b>) Relative movements of the activated and inhibited SH regions. (<b>C</b>) Protein structure (<b>C1</b>) with residues that link the SH to the CD loop (<b>C2</b>). (<b>D</b>) Distance distributions of key residues that drive the channel into the active state. Dark yellow denotes charge–charge interactions with an upper cutoff of 4.0 angstroms. Light green denotes dipole charge interactions with an upper cut off of 4.0 angstroms. (<b>E</b>) Schematic outline of the changes in key residue interactions. (<b>F</b>) Protein structure (<b>F1</b>) with residues that link the SH to the CD loop (<b>F2</b>). (<b>G</b>) Distance distributions of residues that drive the channel into the active state. Dark yellow denotes charge–charge interactions with an upper cutoff of 4.0 angstroms. Pink denotes dipole charge interactions with an upper cutoff of 4.0 angstroms.</p> "> Figure 6
<p>Overview of residue interactions driving the pre-open Apo channel state to the ML297-induced open conformation versus the GAT1587-induced close conformation. (<b>A</b>) A summary of the interaction networks that control channel function located near the compound binding site. (<b>B</b>) Outline of key changes affecting G loop open-state stabilization or a key Lys that coordinates PIP<sub>2</sub> to open the HBC gate.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Sub-Microsecond Long Stochastic Dynamics Simulations Capture Ligand Effects, Validating the Utility of the Computational Model
2.2. Ligand Binding to GIRK1 Subunit of Heterotetramers with GIRK2 or GIRK4 Reveals Key Differences between Residue Conformations
2.3. Ligand Activation Induces a Hydrophobic Chain of Residues in TM1 That Relieves Its Restrain on TM2, Allowing It to Bend and Stabilize the HBC Gate in the Open State
2.4. The TM1 Hydrophobic Chain Regulates K+ Ions at the E141-D173 Acidic Residue Pair of the Permeation Pathway, Depending on the Ligand Bound to the Channel
2.5. Upon ML297 Binding, TM1 Movement Is Transduced to the Slide Helix (SH) and the CD Loop, Freeing GIRK1-K188 Away the GIRK2-E315 (G-Loop) and toward PIP2 Binding
3. Discussion
- Kir channels (characterized by two transmembrane helices -TM1 and TM2- per subunit) utilize TM1 to relieve a restrain of the pore-lining TM2 (of the same subunit type) through the formation of an open-state-dependent TM1 hydrophobic wire (for GIRK1: F87, Y91, F87). These are absolutely or highly conserved residues within Kir channels (the Y91.1 position uses either Tyr or Phe). The ML297-induced hydrophobic wire is further stabilized by TM2 hydrophobic residues of the partner subunit (the other subunit type). This conformational state is communicated to the residue preceding the residue comprising the HBC gate (for GIRK1: M180) (Figure 6A).
- The TM1 hydrophobic wire residues couple to two acidic residues along the permeation pathway. The first, E141.1 at the C-terminal end of the Pore helix (PH), is conserved among GIRK channels (it is a Gln in every other Kir channel). The second, D173 in TM2, is a GIRK1-specific residue (among Kir3 channel subtypes, and Asp, Asn or Glu in every other Kir channel). Ion conduction requires that the PH residue is stabilized by the hydrophobic wire residue while the TM2 acidic residue needs to be freed from interactions with the hydrophobic wire residue (Figure 6A).
- The TM1 movement also repositions the Slide Helix (SH), a 10-aa helical structure arranged parallel to the inner leaflet of the plasma membrane that contains two critical acidic residues (D70 and D77). A CD loop basic residue interacts with the first SH acidic residue (D70) in the presence of the inhibitory ligand but switches to the second one (D77) when the stimulatory ligand binds and the SH moves. This new salt-bridge interaction positions a critical nearby His residue in the CD loop to let go of an important basic residue that controls both gates: the stimulatory ligand frees R313.1 from the GIRK2-H233 and allows to engage the GIRK2-E315 residue away from K188 of GIRK1 stabilizing the GIRK2 G-loop open gate indirectly and allowing the GIRK1-K188 to interact with PIP2, thus stabilizing its HBC open gate directly (Figure 6B).
4. Material and Methods
4.1. Generation of the GIRK1/X Homology Models
4.2. Ligand and Co-Factor Parameterization
4.3. Ligand Docking and Model System Generation
4.4. All-Atom MD Simulations
4.5. Analysis of MD Simulations
4.6. Shortest Pathway Analysis
4.7. Chemical Synthesis
4.8. Electrophysiology
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Gazgalis, D.; Cantwell, L.; Xu, Y.; Thakur, G.A.; Cui, M.; Guarnieri, F.; Logothetis, D.E. Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism. Int. J. Mol. Sci. 2022, 23, 10820. https://doi.org/10.3390/ijms231810820
Gazgalis D, Cantwell L, Xu Y, Thakur GA, Cui M, Guarnieri F, Logothetis DE. Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism. International Journal of Molecular Sciences. 2022; 23(18):10820. https://doi.org/10.3390/ijms231810820
Chicago/Turabian StyleGazgalis, Dimitrios, Lucas Cantwell, Yu Xu, Ganesh A. Thakur, Meng Cui, Frank Guarnieri, and Diomedes E. Logothetis. 2022. "Use of a Molecular Switch Probe to Activate or Inhibit GIRK1 Heteromers In Silico Reveals a Novel Gating Mechanism" International Journal of Molecular Sciences 23, no. 18: 10820. https://doi.org/10.3390/ijms231810820