Mechanism Of Ligand Recognition Research Articles

Calculating Protein-Ligand Residence Times through State Predictive Information Bottleneck Based Enhanced Sampling.

Understanding drug residence times in target proteins is key to improving drug efficacy and understanding target recognition in biochemistry. While drug residence time is just as important as binding affinity, atomic-level understanding of drug residence times through molecular dynamics (MD) simulations has been difficult primarily due to the extremely long time scales. Recent advances in rare event sampling have allowed us to reach these time scales, yet predicting protein-ligand residence times remains a significant challenge. Here we present a semi-automated protocol to calculate the ligand residence times across 12 orders of magnitude of time scales. In our proposed framework, we integrate a deep learning-based method, the state predictive information bottleneck (SPIB), to learn an approximate reaction coordinate (RC) and use it to guide the enhanced sampling method metadynamics. We demonstrate the performance of our algorithm by applying it to six different protein-ligand complexes with available benchmark residence times, including the dissociation of the widely studied anticancer drug Imatinib (Gleevec) from both wild-type Abl kinase and drug-resistant mutants. We show how our protocol can recover quantitatively accurate residence times, potentially opening avenues for deeper insights into drug development possibilities and ligand recognition mechanisms.

Entropy drives the ligand recognition in G-protein-coupled receptor subtypes

Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor's orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.

Structural insights into somatostatin receptor 5 bound with cyclic peptides.

Somatostatin receptor5 (SSTR5) is highly expressed in ACTH-secreting pituitary adenomas and is an important drug target for the treatment of Cushing's disease. Two cyclic SST analog peptides (pasireotide and octreotide) both can activate SSTR5 and SSTR2. Pasireotide is preferential binding to SSTR5 than octreotide, while octreotide is biased to SSTR2 than SSTR5. The lack of selectivity of both pasireotide and octreotide causes side effects, such as hyperglycemia, gastrointestinal disturbance, and abnormal glucose homeostasis. However, little is known about the binding and selectivity mechanisms of pasireotide and octreotide with SSTR5, limiting the development of subtype-selective SST analog drugs specifically targeting SSTR5. Here, we report two cryo-electron microscopy (cryo-EM) structures of SSTR5-Gi complexes activated by pasireotide and octreoitde at resolutions of 3.09 Å and 3.24 Å, respectively. In combination with structural analysis and functional experiments, our results reveal the molecular mechanisms of ligand recognition and receptor activation. We also demonstrate that pasireotide preferentially binds to SSTR5 through the interactions between Tyr(Bzl)/DTrp of pasireotide and SSTR5. Moreover, we find that the Q2.63, N6.55, F7.35 and ECL2 of SSTR2 play a crucial role in octreotide biased binding of SSTR2. Our results will provide structural insights and offer new opportunities for the drug discovery of better selective pharmaceuticals targeting specific SSTR subtypes.

Structure-based characterization and compound identification of the wild-type THF class-II riboswitch.

Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.

Mechanisms of ligand recognition and activation of melanin-concentrating hormone receptors

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 predominately couples to inhibitory G protein, Gi/o, and MCHR2 can only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq at the global resolutions of 3.01 Å and 2.40 Å, respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration when bound to both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation and offer a structural foundation for targeted drug design.

Crystal structure of the complex of CLEC12A and an antibody that interferes with binding of diverse ligands.

C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.

Unveiling the structural mechanisms of nonpeptide ligand recognition and activation in human chemokine receptor CCR8.

The human CC chemokine receptor 8 (CCR8) is an emerging therapeutic target for cancer immunotherapy and autoimmune diseases. Understanding the molecular recognition of CCR8, particularly with nonpeptide ligands, is valuable for drug development. Here, we report three cryo-electron microscopy structures of human CCR8 complexed with Gi trimers in the ligand-free state or activated by nonpeptide agonists LMD-009 and ZK 756326. A conserved Y1.39Y3.32E7.39 motif in the orthosteric binding pocket is shown to play a crucial role in the chemokine and nonpeptide ligand recognition. Structural and functional analyses indicate that the lack of conservation in Y1143.33 and Y1724.64 among the CC chemokine receptors could potentially contribute to the selectivity of the nonpeptide ligand binding to CCR8. These findings present the characterization of the molecular interaction between a nonpeptide agonist and a chemokine receptor, aiding the development of therapeutics targeting related diseases through a structure-based approach.

UNC93B1 variants underlie TLR7-dependent autoimmunity.

UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.

Elucidation of the structural basis for ligand binding and translocation in conserved insect odorant receptor co-receptors

In numerous insects, the olfactory receptor family forms a unique class of heteromeric cation channels. Recent progress in resolving the odorant receptor structures offers unprecedented opportunities for deciphering their molecular mechanisms of ligand recognition. Unexpectedly, these structures in apo or ligand-bound states did not reveal the pathway taken by the ligands between the extracellular space and the deep internal cavities. By combining molecular modeling with electrophysiological recordings, we identified amino acids involved in the dynamic entry pathway and the binding of VUAA1 to Drosophila melanogaster’s odorant receptor co-receptor (Orco). Our results provide evidence for the exact location of the agonist binding site and a detailed and original mechanism of ligand translocation controlled by a network of conserved residues. These findings would explain the particularly high selectivity of Orcos for their ligands.

Ligand recognition and G-protein coupling of trace amine receptor TAAR1.

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Structural basis of hydroxycarboxylic acid receptor signaling mechanisms through ligand binding

Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms.

Ligand-induced activation and G protein coupling of prostaglandin F2α receptor

Prostaglandin F2α (PGF2α), an endogenous arachidonic acid metabolite, regulates diverse physiological functions in many tissues and cell types through binding and activation of a G-protein-coupled receptor (GPCR), the PGF2α receptor (FP), which also is the primary therapeutic target for glaucoma and several other diseases. Here, we report cryo-electron microscopy (cryo-EM) structures of the human FP bound to endogenous ligand PGF2α and anti-glaucoma drugs LTPA and TFPA at global resolutions of 2.67 Å, 2.78 Å, and 3.14 Å. These structures reveal distinct features of FP within the lipid receptor family in terms of ligand binding selectivity, its receptor activation, and G protein coupling mechanisms, including activation in the absence of canonical PIF and ERY motifs and Gq coupling through direct interactions with receptor transmembrane helix 1 and intracellular loop 1. Together with mutagenesis and functional studies, our structures reveal mechanisms of ligand recognition, receptor activation, and G protein coupling by FP, which could facilitate rational design of FP-targeting drugs.

A Biological Perspective of TLR8 Signaling in Host Defense and Inflammation

Abstract Toll-like receptors (TLRs) play important roles in immune responses against pathogens and tumors. Recently, TLR8 has gained attention because of its association with multiple inflammatory diseases, infections and antitumor responses. TLR8 senses the degradation products of single-stranded RNA from microbes and self-released RNA to induce type I interferons, inflammatory gene expression and nucleotide-binding and oligomerization domain–, leucine-rich repeat– and pyrin domain–containing protein 3 (NLRP3) inflammasome activation. So far, the understanding of TLR8 function in vivo is still limited, partially because of lacking a reliable rodent animal model. Murine Tlr8 cannot sense the ligands of human TLR8. In mammals, TLR8 distinguishes live bacteria from dead bacteria to regulate the magnitude of immune responses. Recently, TLR8 has been reported to recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA to induce inflammatory responses, suggesting that TLR8 participates in coronavirus disease 2019 (COVID-19). In this review, we discuss the mechanism of ligand recognition by TLR8, TLR8-mediated signaling pathways and signaling crosstalk between TLR8 and other molecules, and untangle the contribution of TLR8 to inflammatory diseases, infectious diseases, antitumor immunity and vaccination.

Prospect of acromegaly therapy: molecular mechanism of clinical drugs octreotide and paltusotine

Somatostatin receptor 2 (SSTR2) is highly expressed in neuroendocrine tumors and represents as a therapeutic target. Several peptide analogs mimicking the endogenous ligand somatostatin are available for clinical use, but poor therapeutic effects occur in a subset of patients, which may be correlated with subtype selectivity or cell surface expression. Here, we clarify the signal bias profiles of the first-generation peptide drug octreotide and a new-generation small molecule paltusotine by evaluating their pharmacological characteristics. We then perform cryo-electron microscopy analysis of SSTR2-Gi complexes to determine how the drugs activate SSTR2 in a selective manner. In this work, we decipher the mechanism of ligand recognition, subtype selectivity and signal bias property of SSTR2 sensing octreotide and paltusotine, which may aid in designing therapeutic drugs with specific pharmacological profiles against neuroendocrine tumors.

Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4)

Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4–Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

Identification and structural analysis of a carbohydrate-binding module specific to alginate, a representative of a new family, CBM96

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35Å resolution. TM6-N4 exhibited an overall β-sandwich fold architecture with two antiparallel β-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications.

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

Riboswitches are highly conserved RNA elements that located in the 5'-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications.

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2.

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.

A distinctive ligand recognition mechanism by the human vasoactive intestinal polypeptide receptor 2

Class B1 of G protein-coupled receptors (GPCRs) comprises 15 members activated by physiologically important peptide hormones. Among them, vasoactive intestinal polypeptide receptor 2 (VIP2R) is expressed in the central and peripheral nervous systems and involved in a number of pathophysiological conditions, including pulmonary arterial hypertension, autoimmune and psychiatric disorders, in which it is thus a valuable drug target. Here, we report the cryo-electron microscopy structure of the human VIP2R bound to its endogenous ligand PACAP27 and the stimulatory G protein. Different from all reported peptide-bound class B1 GPCR structures, the N-terminal α-helix of VIP2R adopts a unique conformation that deeply inserts into a cleft between PACAP27 and the extracellular loop 1, thereby stabilizing the peptide-receptor interface. Its truncation or extension significantly decreased VIP2R-mediated cAMP accumulation. Our results provide additional information on peptide recognition and receptor activation among class B1 GPCRs and may facilitate the design of better therapeutics.