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Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M₂ muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M₂ receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

G-protein–coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M₂ muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor–nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M₂ receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M₂ receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110–113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.

Adhesion G protein-coupled receptors (ADGRGs) play critical roles in the reproductive, neurological, cardiovascular, and endocrine systems. In particular, ADGRG2 plays a significant role in Ewing sarcoma cell proliferation, parathyroid cell function, and male fertility. In 2022, a cryo-EM structure was reported for the active ADGRG2 bound by an optimized peptide agonist IP15 and the Gs protein. The IP15 peptide agonist was also modified to antagonists 4PH-E and 4PH-D with mutations of the 4PH residue to Glu and Asp, respectively. However, experimental structures of inactive antagonist-bound ADGRs remain to be resolved, and the activation mechanism of ADGRs such as ADGRG2 is poorly understood. Here, we applied Gaussian accelerated molecular dynamics (GaMD) simulations to probe conformational dynamics of the agonist- and antagonist-bound ADGRG2. By performing GaMD simulations, we were able to identify important low-energy conformations of ADGRG2 in the active, intermediate, and inactive states, as well as explore the binding conformations of each peptide. Moreover, our simulations revealed critical peptide-receptor residue interactions during the deactivation of ADGRG2. In conclusion, through GaMD simulations, we uncovered mechanistic insights into peptide (agonist and antagonist) binding and deactivation of the ADGRG2. These findings will potentially facilitate rational design of new peptide modulators of ADGRG2 and other ADGRs.

Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein–ligand binding, especially the drug recognition of GPCRs.

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1 , 2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid–detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine–receptor–G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A 1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein.

G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist-bound state. Here, activation of the M2 receptor is directly observed via accelerated molecular dynamics simulation, in contrast to previous microsecond-timescale conventional molecular dynamics simulations in which the receptor remained inactive. Receptor activation is characterized by formation of a Tyr206 ⁵.⁵⁸–Tyr440 ⁷.⁵³ hydrogen bond and ∼6-Å outward tilting of the cytoplasmic end of transmembrane α-helix 6, preceded by relocation of Trp400 ⁶.⁴⁸ toward Phe195 ⁵.⁴⁷ and Val199 ⁵.⁵¹ and flipping of Tyr430 ⁷.⁴³ away from the ligand-binding cavity. Network analysis reveals that communication in the intracellular domains is greatly weakened during activation of the receptor. Together with the finding that residue motions in the ligand-binding and G-protein-coupling sites of the apo receptor are correlated, this result highlights a dynamic network for allosteric regulation of the M2 receptor activation.

Unraveling the signaling roles of intermediate complexes is pivotal for G protein-coupled receptor (GPCR) drug development. Despite hundreds of GPCR-Gαβγ structures, these snapshots primarily capture the fully activated complex. Consequently, the functions of intermediate GPCR-G protein complexes remain elusive. Guided by a conformational landscape visualized via 19 F quantitative NMR and molecular dynamics (MD) simulations, we determined the structure of an intermediate GPCR-mini-Gα s βγ complex at 2.6 Å using cryo-EM, by blocking its transition to the fully activated complex. Furthermore, we present direct evidence that the complex at this intermediate state initiates a rate-limited nucleotide exchange before transitioning to the fully activated complex. In this state, BODIPY-GDP/GTP based nucleotide exchange assays further indicated the α-helical domain of the Gα is partially open, allowing it to grasp a nucleotide at a non-canonical binding site, distinct from the canonical nucleotide-binding site. These advances bridge a significant gap in our understanding of the complexity of GPCR signaling. Here, the authors employed 19 F NMR and Gaussian accelerated MD simulations to determine the conformational landscape of the adenosine-A2A receptor and mini-Gαsβγ complex, enabling determination of an intermediate structure of the complex with cryoEM.

Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

α 1 -adrenergic receptors (α 1 -ARs) play critical roles in the cardiovascular and nervous systems where they regulate blood pressure, cognition, and metabolism. However, the lack of specific agonists for all α 1 subtypes has limited our understanding of the physiological roles of different α 1 -AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α 1A -AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α 1A -AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α 1A -AR and α 1B -AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α 1A -AR mutants that are not responsive to A61603, and α 1B -AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α 1 -ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists. α1-adrenergic receptors (α1- AR) play critical roles in the cardiovascular and nervous systems. Here, the authors report molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by the agonist A61603.