Sterol transport proteins (STPs) play a pivotal role in cholesterol homeostasis and therefore are essential for healthy human physiology. Despite recent advances in dissecting functions of STPs in the human cell, there is still a significant knowledge gap regarding their specific biological functions and a lack of suitable selective probes for their study. Here, we profile fluorescent steroid-based probes across ten STPs, uncovering substantial differences in their selectivity, aiding the retrospective and prospective interpretation of biological results generated with those probes. These results guided the establishment of an STP screening panel combining diverse biophysical assays, enabling the evaluation of 41 steroid-based natural products and derivatives. Combining this with a thorough structural analysis revealed the molecular basis for STP specific selectivity profiles, leading to the uncovering of several new potent and selective Aster-B inhibitors, and supporting the role of this protein in steroidogenesis.

Neuropeptide Y (NPY), a 36-amino-acid peptide, functions as a neurotransmitter in both the central and peripheral nervous systems by activating the NPY receptor subfamily. Notably, NPY analogs display varying selectivity and exert diverse physiological effects through their interactions with this receptor family. [Pro34]-NPY and [Leu31, Pro34]-NPY, mainly acting on Y1R, reportedly increases blood pressure and postsynaptically potentiates the effect of other vasoactive substances above all, while N-terminal cleaved NPY variants in human body primary mediates angiogenesis and neurotransmitter release inhibition through Y2R. However, the recognition mechanisms of Y1R and Y2R with specific agonists remain elusive, thereby hindering subtype receptor-selective drug development. In this study, we report three cryo-electron microscopy (cryo-EM) structures of Gi2-coupled Y1R and Y2R in complexes with NPY, as well as Y1R bound to a selective agonist [Leu31, Pro34]-NPY. Combined with cell-based assays, our study not only reveals the conserved peptide-binding mode of NPY receptors but also identifies an additional sub-pocket that confers ligand selectivity. Moreover, our analysis of Y1R evolutionary dynamics suggests that this sub-pocket has undergone functional adaptive evolution across different species. Collectively, our findings shed light on the molecular underpinnings of neuropeptide recognition and receptor activation, and they present a promising avenue for the design of selective drugs targeting the NPY receptor family.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease.

Prostaglandin F2α (PGF2α) and thromboxane A2 (TXA2) are endogenous arachidonic acid metabolites, modulating diverse physiological processes including inflammation and cardiovascular homeostasis through activating PGF2α receptor (FP) and TXA2 receptor (TP). Ligands targeting FP and TP have demonstrated efficacy in treating conditions like glaucoma and cardiovascular diseases in humans, as well as reproductive-related diseases in animals. Here, we present five cryoelectron microscopy structures illustrating FP and TP in complex with Gq and bound to PGF2α (endogenous ligand), latanoprost acid (a clinical drug), and two other synthetic agonists. Combined with mutational and functional studies, these structures reveal not only structural features for the specific recognition of endogenous ligands and attainment of receptor selectivity of FP and TP but also the common mechanisms of receptor activation and Gq protein coupling. The findings may enrich our knowledge of ligand recognition and signal transduction of the prostanoid receptor family and facilitate rational ligand design toward these two receptors.

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-β-glucoside-4\'-O-β-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4\'-O-glucosyltransferase (NmF4\'GT) and UDP-glucose:flavone 4\'-O-glucoside 7-O-glucosyltransferase (NmF4\'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4\'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4\'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4\'G7GT. The substrate specificity of NmF4\'GT was examined by comparing its model structure with that of NmF4\'G7GT. The structure of NmF4\'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).

Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms?

Niacin, an age-old lipid-lowering drug, acts through the hydroxycarboxylic acid receptor 2 (HCAR2), a G-protein-coupled receptor (GPCR). Yet, its use is hindered by side effects like skin flushing. To address this, specific HCAR2 agonists, like MK-6892 and GSK256073, with fewer adverse effects have been created. However, the activation mechanism of HCAR2 by niacin and these new agonists is not well understood. Here, we present three cryoelectron microscopy structures of Gi-coupled HCAR2 bound to niacin, MK-6892, and GSK256073. Our findings show that different ligands induce varying binding pockets in HCAR2, influenced by aromatic amino acid clusters (W91ECL1, H1614.59, W1885.38, H1895.39, and F1935.43) from receptors ECL1, TM4, and TM5. Additionally, conserved residues R1113.36 and Y2847.43, unique to the HCA receptor family, likely initiate activation signal propagation in HCAR2. This study provides insights into ligand recognition, receptor activation, and G protein coupling mediated by HCAR2, laying the groundwork for developing HCAR2-targeted drugs.

PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure-function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix-turn-helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases.

Purinergic receptors are membrane proteins that regulate numerous cellular functions by catalyzing reactions involving purine nucleotides or nucleosides. Among the three receptor families, i.e., P1, P2X, and P2Y, the P1 and P2Y receptors share common structural features of class A GPCR. Comprehensive sequence and structural analysis revealed that the P1 and P2Y receptors belong to two distinct groups. They exhibit different ligand-binding site features that can distinguish between specific activators. These specific amino acid residues in the binding cavity may be involved in the selectivity and unique pharmacological behavior of each subtype. In this study, we conducted a structure-based analysis of purinergic P1 and P2Y receptors to identify their evolutionary signature and obtain structural insights into ligand recognition and selectivity. The structural features of the P1 and P2Y receptor classes were compared based on sequence conservation and ligand interaction patterns. Orthologous protein sequences were collected for the P1 and P2Y receptors, and sequence conservation was calculated based on Shannon entropy to identify highly conserved residues. To analyze the ligand interaction patterns, we performed docking studies on the P1 and P2Y receptors using known ligand information extracted from the ChEMBL database. We analyzed how the conserved residues are related to ligand-binding sites and how the key interacting residues differ between P1 and P2Y receptors, or between agonists and antagonists. We extracted new similarities and differences between the receptor subtypes, and the results can be used for designing new ligands by predicting hotspot residues that are important for functional selectivity.

Opioids are effective analgesics, but their use is beset by serious side effects, including addiction and respiratory depression, which contribute to the ongoing opioid crisis. The human opioid system contains four opioid receptors (μOR, δOR, κOR, and NOPR) and a set of related endogenous opioid peptides (EOPs), which show distinct selectivity toward their respective opioid receptors (ORs). Despite being key to the development of safer analgesics, the mechanisms of molecular recognition and selectivity of EOPs to ORs remain unclear. Here, we systematically characterize the binding of EOPs to ORs and present five structures of EOP-OR-Gi complexes, including β-endorphin- and endomorphin-bound μOR, deltorphin-bound δOR, dynorphin-bound κOR, and nociceptin-bound NOPR. These structures, supported by biochemical results, uncover the specific recognition and selectivity of opioid peptides and the conserved mechanism of opioid receptor activation. These results provide a structural framework to facilitate rational design of safer opioid drugs for pain relief.