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Cited by 7Receptor Mechanisms and SignalingProtein Structure and DynamicsComputational Drug Discovery Methods

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Hidden GPCR structural transitions addressed by multiple walker supervised molecular dynamics (mwSuMD)

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Deganutti, Giuseppe, Pipito, Ludovico, Rujan, Roxana Maria, Weizmann, Tal, Griffin, Peter, Ciancetta, Antonella, Moro, Stefano, Reynolds, Christopher Arthur·2025·eLife
DOI 10.7554/elife.96513PMC PMC12043319

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Abstract

The structural basis for the pharmacology of human G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra- and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V 2 . Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and G s -bound state, and the guanosine diphosphate (GDP) release from G s . To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.

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Figures

Figure 1. Arginine vasopressin (AVP) supervised molecular dynamics (SuMD) and multiple walker SuMD (mwSuMD) binding simulations to vasopressin 2 receptor (V2R) (100 ps time windows). (a) Chemical structure and (b) binding more within the V2R orthosteric binding site; AVP is represented in orange stick, while V2R is in white ribbon and gray stick. For each set of settings (c–f) the root mean square deviation (RMSD) of AVP Cα atoms to the cryo-electron microscopy (cryo-EM) structure 7DW9 is reported during the time course of each SuMD (c) or mwSuMD (d–f) replica alongside the RMSD values distribution and the snapshot corresponding to the lowest RMSD values (AVP from the cryo-EM structure 7DW9 is in a cyan stick representation, while AVP from simulations is in a tan stick representation). A complete description of the simulation settings is reported in Table 1 and the Methods section. The dashed red line indicates the AVP RMSD during a classic (unsupervised) equilibrium MD simulation of the X-ray AVP:V2R complex (Figure 1—figure supplement 1).
Figure 1. Arginine vasopressin (AVP) supervised molecular dynamics (SuMD) and multiple walker SuMD (mwSuMD) binding simulations to vasopressin 2 receptor (V2R) (100 ps time windows). (a) Chemical structure and (b) binding more within the V2R orthosteric binding site; AVP is represented in orange stick, while V2R is in white ribbon and gray stick. For each set of settings (c–f) the root mean square deviation (RMSD) of AVP Cα atoms to the cryo-electron microscopy (cryo-EM) structure 7DW9 is reported during the time course of each SuMD (c) or mwSuMD (d–f) replica alongside the RMSD values distribution and the snapshot corresponding to the lowest RMSD values (AVP from the cryo-EM structure 7DW9 is in a cyan stick representation, while AVP from simulations is in a tan stick representation). A complete description of the simulation settings is reported in Table 1 and the Methods section. The dashed red line indicates the AVP RMSD during a classic (unsupervised) equilibrium MD simulation of the X-ray AVP:V2R complex (Figure 1—figure supplement 1).CC BY · PubMed Central
Figure 1—figure supplement 1. Classic molecular dynamics (MD) simulation of the AVP:V2R complex (PDB ID 7DW9). The average AVP root mean square deviation (RMSD) (computed on the Cα atoms) after stabilization of the system (second half of the trajectory) is indicated. AVP, arginine vasopressin; V2R, vasopressin 2 receptor.
Figure 1—figure supplement 1. Classic molecular dynamics (MD) simulation of the AVP:V2R complex (PDB ID 7DW9). The average AVP root mean square deviation (RMSD) (computed on the Cα atoms) after stabilization of the system (second half of the trajectory) is indicated. AVP, arginine vasopressin; V2R, vasopressin 2 receptor.CC BY · PubMed Central
Figure 1—figure supplement 2. Arginine vasopressin (AVP) supervised molecular dynamics (SuMD) and multiple walker SuMD (mwSuMD) binding simulations to vasopressin 2 receptor (V2R) (600 ps time windows). For each set of settings (a-e), the RMSD of AVP Cα atoms to the cryo-EM structure 7DW9 is reported during the time course of each SuMD (a) or mwSuMD (b-e) replica, alongside the RMSD values distribution and the snapshot corresponding to the lowest RMSD (AVP from the cryo EM structure 7DW9 is in a cyan stick representation, while AVP from simulations is in a tan stick representation). A complete description of the simulation settings is reported in Table 1 and the Methods section. The dashed red line indicates the AVP RMSD during a classic (unsupervised) equilibrium MD simulation of the X-ray AVP:V2R complex (Figure 1—figure supplement 1).
Figure 1—figure supplement 2. Arginine vasopressin (AVP) supervised molecular dynamics (SuMD) and multiple walker SuMD (mwSuMD) binding simulations to vasopressin 2 receptor (V2R) (600 ps time windows). For each set of settings (a-e), the RMSD of AVP Cα atoms to the cryo-EM structure 7DW9 is reported during the time course of each SuMD (a) or mwSuMD (b-e) replica, alongside the RMSD values distribution and the snapshot corresponding to the lowest RMSD (AVP from the cryo EM structure 7DW9 is in a cyan stick representation, while AVP from simulations is in a tan stick representation). A complete description of the simulation settings is reported in Table 1 and the Methods section. The dashed red line indicates the AVP RMSD during a classic (unsupervised) equilibrium MD simulation of the X-ray AVP:V2R complex (Figure 1—figure supplement 1).CC BY · PubMed Central
Figure 2. Multiple walker supervised molecular dynamics (mwSuMD) (un)binding simulations of arginine vasopressin (AVP). Vasopressin 2 receptor (V2R) residues involved in mwSuMD simulations of AVP. (a) Binding (dynamic docking); (b) unbinding simulations. (c) Dissociation of AVP from V2R. The root mean square deviation (RMSD) of AVP to the initial bound state is reported during the time course of five replicas of SuMD and mwSuMD, respectively.
Figure 2. Multiple walker supervised molecular dynamics (mwSuMD) (un)binding simulations of arginine vasopressin (AVP). Vasopressin 2 receptor (V2R) residues involved in mwSuMD simulations of AVP. (a) Binding (dynamic docking); (b) unbinding simulations. (c) Dissociation of AVP from V2R. The root mean square deviation (RMSD) of AVP to the initial bound state is reported during the time course of five replicas of SuMD and mwSuMD, respectively.CC BY · PubMed Central
Figure 3. Multiple walker supervised molecular dynamics (mwSuMD) simulation of PF06882961 binding to glucagon-like peptide-1 receptor (GLP-1R) and receptor activation. Each panel reports the root mean square deviation (RMSD) to the position of the ligand in the active state (top panel) or a GLP-1R structural element over the time course (all but ECL3 converging to the active state). ECD: extracellular domain; TM: transmembrane helix; ECL: extracellular loop. The mwSuMD simulation was performed with four different settings over 1 µs in total. The red dashed lines show the initial RMSD value for reference.
Figure 3. Multiple walker supervised molecular dynamics (mwSuMD) simulation of PF06882961 binding to glucagon-like peptide-1 receptor (GLP-1R) and receptor activation. Each panel reports the root mean square deviation (RMSD) to the position of the ligand in the active state (top panel) or a GLP-1R structural element over the time course (all but ECL3 converging to the active state). ECD: extracellular domain; TM: transmembrane helix; ECL: extracellular loop. The mwSuMD simulation was performed with four different settings over 1 µs in total. The red dashed lines show the initial RMSD value for reference.CC BY · PubMed Central
Figure 3—figure supplement 1. Multiple walker supervised molecular dynamics (mwSuMD) simulation of PF06882961, glucagon-like peptide-1 receptor (GLP-1R) activation, and guanosine diphosphate (GDP) release. (a) PF06882961 binding site in the apo and holo GLP-1R. In the apo GLP-1R (left transparent ribbon) the residues forming the binding site of the NPA PF06882961 are scattered due to the different conformation of the receptor. (b, c) NPAs stabilize divergent GLP-1R active conformations: (a) Different orientations of TM2, ECL1, and TM3; (b) divergent conformations of extracellular domain (ECD), ECL3, and TM7; (c) interaction fingerprint of NPAs (TT-OAD2, CHU-128, and PF06882911) and the endogenous agonist peptide GLP-1. Red bars indicate interactions with distinct residues in the GLP-1R.
Figure 3—figure supplement 1. Multiple walker supervised molecular dynamics (mwSuMD) simulation of PF06882961, glucagon-like peptide-1 receptor (GLP-1R) activation, and guanosine diphosphate (GDP) release. (a) PF06882961 binding site in the apo and holo GLP-1R. In the apo GLP-1R (left transparent ribbon) the residues forming the binding site of the NPA PF06882961 are scattered due to the different conformation of the receptor. (b, c) NPAs stabilize divergent GLP-1R active conformations: (a) Different orientations of TM2, ECL1, and TM3; (b) divergent conformations of extracellular domain (ECD), ECL3, and TM7; (c) interaction fingerprint of NPAs (TT-OAD2, CHU-128, and PF06882911) and the endogenous agonist peptide GLP-1. Red bars indicate interactions with distinct residues in the GLP-1R.CC BY · PubMed Central
Figure 3—figure supplement 2. Dynamic feautures of GLP-1R activation and Gs binding. (a) Rotation of TM6 during glucagon-like peptide-1 receptor (GLP-1R) activation simulated by multiple walker supervised molecular dynamics (mwSuMD). The angle was measured as the dihedral formed by the backbone alpha carbon atoms T353, I357, T362, and I366. The angle values of the inactive and active cryo-electron microscopy (cryo-EM) structures are reported as references; (b) PF06882961 MM-GBSA binding energy during Gs binding; (d) guanosine diphosphate (GDP) MM-GBSA binding energy during Gs binding.
Figure 3—figure supplement 2. Dynamic feautures of GLP-1R activation and Gs binding. (a) Rotation of TM6 during glucagon-like peptide-1 receptor (GLP-1R) activation simulated by multiple walker supervised molecular dynamics (mwSuMD). The angle was measured as the dihedral formed by the backbone alpha carbon atoms T353, I357, T362, and I366. The angle values of the inactive and active cryo-electron microscopy (cryo-EM) structures are reported as references; (b) PF06882961 MM-GBSA binding energy during Gs binding; (d) guanosine diphosphate (GDP) MM-GBSA binding energy during Gs binding.CC BY · PubMed Central
Figure 4. Glucagon-like peptide-1 receptor (GLP-1R) key structural motifs during multiple walker supervised molecular dynamics (mwSuMD) GLP-1R activation. (a) Root mean square deviation (RMSD) to the active state GLP-1R (7LCI) of the residues forming the central polar network (magenta) and TM6 kink (orange) during mwSuMD of receptor activation; at the end of the simulations minimum values were reached (dashed square). (b) The position of the polar network within the core of transmembrane domain (TMD) (left-hand panel) and comparison between inactive, active, and mwSuMD final states for the side chains of the residues forming of the polar network. (c) The position of the TM6 kink (right-hand panel) and comparison between inactive, active, and mwSuMD final states for the side chains of the residues forming the TM6 kink.
Figure 4. Glucagon-like peptide-1 receptor (GLP-1R) key structural motifs during multiple walker supervised molecular dynamics (mwSuMD) GLP-1R activation. (a) Root mean square deviation (RMSD) to the active state GLP-1R (7LCI) of the residues forming the central polar network (magenta) and TM6 kink (orange) during mwSuMD of receptor activation; at the end of the simulations minimum values were reached (dashed square). (b) The position of the polar network within the core of transmembrane domain (TMD) (left-hand panel) and comparison between inactive, active, and mwSuMD final states for the side chains of the residues forming of the polar network. (c) The position of the TM6 kink (right-hand panel) and comparison between inactive, active, and mwSuMD final states for the side chains of the residues forming the TM6 kink.CC BY · PubMed Central
Figure 5. Glucagon-like peptide-1 receptor (GLP-1R) activation and Gs binding. (a–d) Sequence of simulated events during the multiple walker supervised molecular dynamics (mwSuMD) Gs:GLP-1R simulations. (e) Root mean square deviation (RMSD) of Gsα to the experimental GLP-1R:Gs complex (PDB 7LCJ) during three mwSuMD replicas; the RMSD to the experiential bound conformation (7LCJ) during the second part of Replica 2 (red dashed line) is reported for each Gs subunit. RMSDs were computed on Gα residues 11–43 and 205–394 to the experimental structure 7LCI after superimposition on GLP-1R residues 140–240 Cα atoms. (f) RMSD of the αG-α4 loop (purple), the distance between K3426.31 and D323Gα (salmon), and the distance between guanosine diphosphate (GDP) and D295Gα (green) during Gs binding, alpha-helical domain (AHD) opening and GDP dissociation; (g) and (h) comparison between states extracted from before and after AHD opening. Before AHD opening (a), GLP-1R ICL3 interacted with D323Gα and D295Gα interacted with GDP; after AHD opening (b) and αG-α4 loop reorganization (curved red arrow), αG and D295Gα moved away from GDP (straight red arrow), destabilizing its binding to Gs.
Figure 5. Glucagon-like peptide-1 receptor (GLP-1R) activation and Gs binding. (a–d) Sequence of simulated events during the multiple walker supervised molecular dynamics (mwSuMD) Gs:GLP-1R simulations. (e) Root mean square deviation (RMSD) of Gsα to the experimental GLP-1R:Gs complex (PDB 7LCJ) during three mwSuMD replicas; the RMSD to the experiential bound conformation (7LCJ) during the second part of Replica 2 (red dashed line) is reported for each Gs subunit. RMSDs were computed on Gα residues 11–43 and 205–394 to the experimental structure 7LCI after superimposition on GLP-1R residues 140–240 Cα atoms. (f) RMSD of the αG-α4 loop (purple), the distance between K3426.31 and D323Gα (salmon), and the distance between guanosine diphosphate (GDP) and D295Gα (green) during Gs binding, alpha-helical domain (AHD) opening and GDP dissociation; (g) and (h) comparison between states extracted from before and after AHD opening. Before AHD opening (a), GLP-1R ICL3 interacted with D323Gα and D295Gα interacted with GDP; after AHD opening (b) and αG-α4 loop reorganization (curved red arrow), αG and D295Gα moved away from GDP (straight red arrow), destabilizing its binding to Gs.CC BY · PubMed Central
Figure 5—figure supplement 1. G protein binding simulations to β2 adrenoreceptor (β2 AR) and A1 receptor (A1R). (a) Root mean square deviation (RMSD) of Gsα to the experimental complex (PDB 3NS6) during three multiple walker supervised molecular dynamics (mwSuMD) replicas; (b) RMSD of Gsβ to the experimental complex (PDB 3NS6) during three mwSuMD replicas; (c) superposition of the experimental Gs:β2 AR complex (transparent ribbon) and the MD frame with the lowest Gsα RMSD (3.94 Å); (d) RMSD of Giα (residues 243–355) to the A1R experimental complex (PDB 6D9H) during an mwSuMD simulation (red, magnified in the box) and a 1000-ns-long classic MD simulation (black); (e) two-view superposition of the experimental Gi:A1R complex (transparent ribbon) and the MD frame with the lowest Giα RMSD (4.82 Å).
Figure 5—figure supplement 1. G protein binding simulations to β2 adrenoreceptor (β2 AR) and A1 receptor (A1R). (a) Root mean square deviation (RMSD) of Gsα to the experimental complex (PDB 3NS6) during three multiple walker supervised molecular dynamics (mwSuMD) replicas; (b) RMSD of Gsβ to the experimental complex (PDB 3NS6) during three mwSuMD replicas; (c) superposition of the experimental Gs:β2 AR complex (transparent ribbon) and the MD frame with the lowest Gsα RMSD (3.94 Å); (d) RMSD of Giα (residues 243–355) to the A1R experimental complex (PDB 6D9H) during an mwSuMD simulation (red, magnified in the box) and a 1000-ns-long classic MD simulation (black); (e) two-view superposition of the experimental Gi:A1R complex (transparent ribbon) and the MD frame with the lowest Giα RMSD (4.82 Å).CC BY · PubMed Central
Figure 5—figure supplement 2. Comparison of the inactive (6EG8, white), nucleotide-free (7LCI, black), and guanosine diphosphate (GDP)-dissociated (multiple walker supervised molecular dynamics [mwSuMD] simulation, green) β6-α5 loop. Bound (white, 6EG8) and dissociated (green, mwSuMD) GDP is shown as a stick representation.
Figure 5—figure supplement 2. Comparison of the inactive (6EG8, white), nucleotide-free (7LCI, black), and guanosine diphosphate (GDP)-dissociated (multiple walker supervised molecular dynamics [mwSuMD] simulation, green) β6-α5 loop. Bound (white, 6EG8) and dissociated (green, mwSuMD) GDP is shown as a stick representation.CC BY · PubMed Central
Figure 6. Preparation to GLP-1R mwSuMD simulations. (a) Potential energy surface derived from the scan of the PF06882961 rotatable bond through which the dihedrals NG2R51-CG321-CG3C41-CG3C41 (penalty = 143.5) and NG2R51-CG321-CG3C41-OG3C51 (penalty = 152.4) pass. The curve obtained from the original CGenFF parameters (red) was optimized (blue) to resemble the energy profile from quantum-mechanics computations at the HF/6-31g(d) level of theory (black). (b) Distances between glucagon-like peptide-1 receptor (GLP-1R) key residues E1381.33 and W214ECL1 (black), H3636.52 and F3907.45 (blue) and Q47ECD and S310ECL2 (red) during 8 μs of classic molecular dynamics (MD); (c) positions of the three couples of residues reported as two-side views.
Figure 6. Preparation to GLP-1R mwSuMD simulations. (a) Potential energy surface derived from the scan of the PF06882961 rotatable bond through which the dihedrals NG2R51-CG321-CG3C41-CG3C41 (penalty = 143.5) and NG2R51-CG321-CG3C41-OG3C51 (penalty = 152.4) pass. The curve obtained from the original CGenFF parameters (red) was optimized (blue) to resemble the energy profile from quantum-mechanics computations at the HF/6-31g(d) level of theory (black). (b) Distances between glucagon-like peptide-1 receptor (GLP-1R) key residues E1381.33 and W214ECL1 (black), H3636.52 and F3907.45 (blue) and Q47ECD and S310ECL2 (red) during 8 μs of classic molecular dynamics (MD); (c) positions of the three couples of residues reported as two-side views.CC BY · PubMed Central

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Research works printed but not linked: 1

  1. Pipitò L. 2025. mwSuMD - Molecular Dynamics Supervision Tool. Software Heritage. swh:1:rev:b5cd2dacea7b003df96ee88a34919d87b937b744. https://archive.softwareheritage.org/swh:1:dir:3a780e7322852c01cf6ca71a61a2b161b5ccda6c;origin=https://github.com/pipitoludovico/mwSuMD_multiEngine;visit=swh:1:snp:17cec8ea7b03cfd8e5c4cfddc542c49b3796d350;anchor=swh:1:rev:b5cd2dacea7b003df96ee88a34919d87b937b744.

Metadata

Authors
Deganutti, G., Pipito, L., Rujan, R. M., Weizmann, T., Griffin, P., Ciancetta, A., Moro, S., & Reynolds, C. A.
Affiliations
  • Coventry University (GB)
  • Centre for Health and Life Sciences, Coventry University
  • University of Ferrara (IT)
  • University of Padua (IT)
  • University of Essex (GB)
Corresponding author
  • Deganutti, Giuseppe
  • Reynolds, Christopher Arthur
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2025-04-30
Volume
13
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.96513
PMC
PMC12043319
OpenAlex
W4396889854
Cited by
7
References
111
Citations by year
2026: 4 · 2025: 3
Type
posted content
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Pure (Coventry University) (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Biotechnology and Biological Sciences Research Council (BB/W016974/1)
  • Diabetes UK (BDA 20/0006307)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Drug Discovery › Computational Drug Discovery Methods
Keywords
G protein-coupled receptors, adaptive sampling, structural phase transition, V2 receptor, GLP-1 receptor, receptor activation, Gs protein, GDP release, receptor-G protein coupling, metastable states
MeSH terms
Glucagon-Like Peptide-1 Receptor, Algorithms, Humans, Protein Binding, Protein Conformation, Vasopressins, Receptors, Vasopressin, Receptors, G-Protein-Coupled, Molecular Dynamics Simulation

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