Duy Phuoc Tran

Taste receptor type 1 (TAS1R), which consists of sweet and umami receptors in humans, senses nutrients (such as sugars and amino acids) with substrate specificity that is often broad and varies among animals and subtypes. However, the structural basis for achieving diverse specificities remains largely elusive. Here, we present the crystal structure of the ligand-binding domain (LBD) of Tas1r1/Tas1r3 heterodimer from pufferfish, an ortholog of the human umami taste receptor. The overall structure of Tas1r1/Tas1r3LBD resembles previously reported TAS1R structures, indicating a conserved core architecture within the family. Nevertheless, pufferfish Tas1r1/Tas1r3 was found to bind and respond to both l - and d -amino acids, even though TAS1Rs are considered to respond to either enantiomer. Structural and mutational analyses revealed that this nonrigorous stereochemical recognition is attributed to intersubdomain interactions that latch the cleft containing the amino acid-binding site. These interactions prevent the cleft from fully opening, thereby stabilizing the active conformation, even if the ligand has a different chirality. These results suggest that different substrate specificities in TAS1Rs can be acquired not only by the gain or loss of direct interactions with the substrate but also by the gain of intramolecular interactions, which alter the conformational equilibrium of the receptor.

GPR84 is an orphan class A GPCR primarily expressed in immune cells, where it plays key roles in inflammation and metabolism. Here, we present the cryo-electron microscopy structures of the GPR84-Gi complex bound to the G protein–biased agonist DL-175, and the inactive state of GPR84 bound to the antagonist GLPG1205. Combined with signaling assays and molecular dynamics simulations, these structures elucidate the conformational landscape spanning the inactive and G protein-biased active states of GPR84, providing a mechanistic basis for biased agonism. Notably, steric interactions between DL-175 and L336^6.52 selectively preclude the conformational changes required for efficient β-arrestin recruitment without compromising G protein activation. These structural insights provide a structural context for the rational design of GPR84-targeted therapeutics with precisely tuned signaling profiles.

G-protein-coupled receptors (GPCRs) are essential mediators of cellular processes, and their dysfunction is implicated in various diseases. Although pharmacological approaches are powerful for elucidating GPCR functions, careful consideration of potential off-target effects remains crucial. Here, we present an off-target-free chemogenetic platform to dissect the physiological roles of target GPCRs. As a proof-of-concept, we focused on the adenosine A _2A receptor (A _2A R), a prototypical GPCR involved in neurodegenerative diseases. Leveraging the ligand recognition mechanisms of A _2A R, we designed a prominent chemogenetic pair comprising a clinical A _2A R antagonist and an engineered A _2A R mutant that is insensitive to the antagonist but retains responsiveness to adenosine. Using this pair, we uncovered the critical role of A _2A R in neurite outgrowth under hypoxic conditions in neuron-like cells, while avoiding potential off-target effects. Furthermore, we demonstrate the generalizability of this chemogenetic approach to other class A GPCRs, offering a versatile platform for receptor-specific dissection of GPCR signaling.

In vitro evolution is one of the powerful methods for altering the characteristics of a protein of interest; however, its application to membrane proteins remains challenging due to the requirement of a membrane environment in vitro. This study presents the first successful in vitro evolution of human adenosine A_2A receptor (A_2AR), one of the G protein-coupled receptors (GPCRs), achieved through combining cell-free protein synthesis (CFPS), a nanodisc for membrane mimicry, and a ribosome display. A saturation mutagenesis library of A_2AR targeting the L167 and L267 residues, a library with a diversity of only 400, was constructed without sequence bias. Three rounds of affinity selection against A_2AR-selective antagonist ZM241385 enriched the L167N/L267A and L167N/L267S mutants. These mutants exhibited greater than 10-fold improved sensitivity to ZM241385 in mammalian cell-based assays while retaining responsiveness to the endogenous ligand adenosine. Molecular dynamics simulations revealed new interactions between ZM241385 and A_2AR mutants that are likely to contribute to stabilizing the conformation of the complexes. Finally, as an example of an application, the obtained mutant was used for cell-type-specific inhibition of A_2AR signaling by ZM241385. This study demonstrates a powerful strategy for engineering GPCRs entirely in vitro with tailored ligand-binding properties, even from a library with small diversity, and shows that the evolved mutants exhibit improved functionality in mammalian cells.

The human adenosine A _2A receptor (A _2A R) is a prototypical member of the class A family of G Protein–Coupled Receptors (GPCRs), which are engaged by over one-third of FDA-approved drugs. Here, we used ¹⁹ F NMR to simultaneously evaluate functional states from the perspective of a CF ₃ -tag on transmembrane helix-6 (TM6) and all 5-fluorotryptophan reporters. Whereas spectra from the TM6 tag reveal a dynamic conformational ensemble, the tryptophan spectra exhibit more discrete ligand-dependent states. These ligand-dependent signatures provide insights into microswitches including a universal toggle switch which interfaces with a sodium binding pocket. Over 560 GPCRs share this toggle switch — sodium pocket cluster, implying a fundamental role in activation. Computational rigidity-theory reveals tryptophan reporters reside along prominent allosteric activation pathways, underscoring their roles in mechanical signal propagation and corroborating NMR observations. Among them, W246 ^6.48 , associated with the toggle switch, plays a critical role in regulation of allosteric networks spanning the orthosteric pocket and extending via tryptophan-rich pathways through the receptor to the A _2A R-Gβ interface and the nucleotide pocket in G _s α. While higher sodium concentrations (~100 mM) predictably stabilize the inactive conformation of the receptor, lower concentrations (below 40 mM) greatly enhanced the presence of the activation ensemble and in particular, the precoupled state, suggesting that the release of sodium from the conserved pocket—either through basal conditions for the apo receptor, or upon binding of the agonist—enables sampling of a precoupled state, ultimately needed to initiate activation and coupling.

Understanding brain function requires tools that allow precise manipulation of receptor signaling in defined cell types within intact neural circuits. Optogenetics and conventional chemogenetic approaches primarily enable cell-type-specific control of neuronal excitability using engineered receptors or ion channels. However, direct and reversible inhibition of endogenous neurotransmitter receptors in defined cell types has remained technically inaccessible. Here, we introduce native receptor-targeted chemogenetics (NARCH), a chemogenetic strategy that integrates structure-guided receptor engineering with allosteric ligand design to achieve reversible and temporally precise inhibition of endogenous receptor signaling with cell-type specificity in vivo . By applying NARCH to metabotropic glutamate receptor 1 (mGlu1), we demonstrate that mGlu1 signaling in cerebellar Purkinje cells is required for stabilization of motor learning across training sessions in freely moving mice. NARCH thus establishes a receptor-level chemogenetic framework for causal analysis of neural circuits and behavior.

With the recent extensive improvement of classical biomolecular force fields in solution, the performance of these in vacuum simulations remains unclear. In this work, we perform extensive replica exchange molecular dynamics (REMD) to compare the recent modern force fields. Therefore, we investigate the sampling conformations of the force fields and perform quantum mechanical total energy calculations. We found that the treatment of CMAP or dihedral angle functions does not strongly affect the sampling of the force fields in vacuum simulations, but the inter-residue mainchain-side chain interaction may play important roles. In addition, the sampling area in PC space and the radius of gyration versus N-terminus to C-terminus distances are not dominantly the highest in all cases using ff14SB, but are cumulatively the highest across the 9 peptides in this work. This information may help support the development of future universal transferable force fields.

Post-translational modifications play important roles in the regulation of protein function, with N^ε acetylation being a key reversible modification affecting processes such as transcription, metabolism, and stress responses. Sirtuins, particularly SIRT1 and its ortholog, Sir2, are NAD+-dependent deacetylases that target both histone and nonhistone proteins, including the tumor suppressor p53. Acetylation of p53 on K382 influences its degradation and transcriptional activity. Despite structural studies of the Sir2/acetylated p53 complex, the role of the conserved cofactor-binding loop (CBL) in regulating NAD+ binding and deacetylation remains unclear. Using both conventional molecular dynamics (MD) and parallel cascade selection MD (PaCS-MD) simulations, we investigated the conformational dynamics of the Sir2/acetylated and nonacetylated p53 complexes, focusing on the conformational changes in the CBL of Sir2 in response to p53 acetylation. We identified open and closed states of the NAD+ binding pocket caused by CBL conformational changes depending on p53 acetylation and deacetylation. The forward allosteric effect of the acetylated p53 binding was found to open the NAD+-binding pocket, which is expected to promote NAD+ binding. In contrast, the binding of nonacetylated p53 is significantly weaker, and the reverse allosteric effect drives the pocket closure. These sequential allosteric effects positively accelerate the reaction cycle, which can be considered a "tandem allostery of the reactant (acetylated p53) and the product (deacetylated p53)". Combining these simulations with entropy transfer analysis, K382 was found to initiate multiple communication routes through strands β7 and β9, and the FEG loop, ultimately converging on the CBL via the helical small domain, the Rossmann-fold domain, or directly from p53, thereby highlighting the critical role of CBL in NAD+ binding and p53 deacetylation.