The SIMDA framework maps PIP2-binding sites regulating the KCNQ1 channel

Figures

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  Fig. 1. The Systems investigated and SIMDA pipeline. a Superimposed conformations of 6V00 and 6V01 monomers. b Intact tetrameric conformation of 6V00. c Schematic of the SIMDA framework for studying protein-lipid binding. d, e The framework analyzes lipid dynamics, thermodynamics, and binding sites through a stepwise approach: Stepwise Integrated Multi-scale Dynamics begins with structure preparation. CG-MD simulations identify lipid binding sites (e.g., Bsid0, Bsid1, Bsidn), which are refined via AA-MD. Wt-MetaD enables enhanced sampling of dissociation pathways. Advanced Analysis includes binding site screening based on interaction stability, UMAP-Leiden clustering to identify functionally equivalent sites, interaction analysis using hydrogen bond analysis and MM/GBSA, and dissociation trajectory analysis for thermodynamic and kinetic insights.

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  Fig. 2. Identification and Screening of PIP2 binding sites based on CG -MD and AA-MD simulations. a 2D-density maps in the plane of the bilayer (XY) of the distribution of PIP2 relative to the four inactive state systems: KCNQ1, KCNQ1-KCNE3, KCNQ1-CaM, and KCNQ1-KCNE3-CaM. b Density maps of PIP2 binding across the Active state of KCNQ1, KCNQ1-KCNE3, KCNQ1-CaM, and KCNQ1-KCNE3-CaM. c Binding sites identified and screened from the AA-MD simulations in the Active KCNQ1-KCNE3-CaM system were subjected to the second screening selection. Moreover, PIP2 binding at the excluded site Bsid10 was found to be unstable within the first 100 ns, as evidenced by the 300 ns AA-MD simulations (3000 frames in total).

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  Fig. 3. 6 PIP2 binding sites on KCNQ1. a The classifications of binding modes of PIP2 at the 6 binding sites. b Binding modes of PIP2 at the 6 binding sites. c Superposition of experimental structures and MD simulation results for four KCNQ channel complexes: KCNQ1 (PDB: 6V01, C1 site), KCNQ5 (PDB: 9LIZ, C2 site), KCNQ4 (PDB: 7VNP, C3 site), and KCNQ1 (PDB: 9WD8, C1 and C3 sites). Experimental protein structures are shown in white cartoon representation. PIP2 molecules resolved in the cryo-EM structures are displayed as cyan spheres. Simulation-derived channel subunits are highlighted in green (C1), purple (C2), and light blue (C3) cartoons, with corresponding simulation-derived PIP2 molecules shown in matching colors. d Different classifications and numbers of PIP2 binding sites in the Inactive and Active KCNQ1 systems.

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  Fig. 4. Contact, hydrogen bond, and MM/GBSA energy decomposition analyses of hotspot residues based on AA-MD simulations. a Distribution of PIP2 binding sites in the Inactive and Active KCNQ1 systems, respectively. The numbers in red represent different binding sites (C0-5). b Contact, H-bond, and MM/GBSA energy decomposition occupancy between hotspot residues and PIP2 for each binding site cluster, ranging from 0% to 40%. Orange represents representative contact frequency, cyan represents representative H-bond frequency, and green represents energy decomposition frequency.

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  Fig. 5. The dissociation pathways of PIP2 at the binding sites C1-Free, C1-KCNE3, and C1-CaM based on Wt-MetaD simulations. a–c FES during the dissociation process of PIP2 at C1-Free, C1-KCNE3, and C1-CaM Systems; Distance fluctuations between the S2-S3 linker, S2, S3, and S4 regions near the C-terminal and their positions in the initial simulation frame for C1-Free; Principal Component Analysis (PCA) of Key Regions (S2-S3 Linker, S2, S3, S4, and S4-S5 Linker) in the C1-Free, C1-KCNE3, and C1-CaM Systems; Conformational changes near the C-terminal region, including the S2-S3 linker, S2, S3, S4, and S4-S5 linker, during transitions between key stable and metastable states for C1-Free, C1-KCNE3, and C1-CaM Systems.

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  Fig. 6. Overview of the dynamic binding and dissociation behavior of PIP2 across multiple sites, as characterized by Wt-MetaD simulations. a Representative dynamic circular pathways of binding-site transfers during the PIP2 binding and dissociation process. b Transparent points denote distinct categories of binding sites. In each subplot (Path1-8), dissociation trajectories obtained from metadynamics simulations are illustrated, where spheres transition from blue to red, indicating the temporal progression of PIP2 positions during unbinding.