The 3-hydroxyacyl-CoA dehydratase 1/2 form complex with trans-2-enoyl-CoA reductase involved in substrates transfer in very long chain fatty acid elongation.

Very long-chain fatty acids (VLCFAs) are fatty acids with a carbon chain length greater than 18 carbons (>C18) and exhibit various functions, such as in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function, and anti-inflammation. VLCFAs are absorbed by dietary or elongated from endogenous hexadecanoyl acids (C16). Similar to long-chain fatty acid synthesis, VLCFAs elongation begins with acyl-CoA and malonyl-CoA as sources, and the length of the acyl chain is extended by two carbon units in each cycle. However, the VLCFAs elongation machinery is located in ER membrane and consists of four components, FA elongase (ELOVL), 3-ketoacyl-CoA reductase (KAR), 3-hydroxyacyl-CoA dehydratase (HACD), and trans-2-enoyl-CoA reductase (TECR), which is different with the long-chain fatty acid machinery fatty acid synthase (FAS) complex. Although the critical components in the elongation cycle are identified, the detailed catalytic and regulation mechanisms are still poorly understood. Here, we focused on the structural and biochemical analysis of TECR-associated VLCFA elongation reactions. Firstly, we identified a stable complex of human HACD2-TECR based on extensive in vitro characterizations. Combining computational modeling and biochemical analysis, we confirmed the critical interactions between TECR and HACD1/2. Then, we proposed the putative substrate binding sites and catalytic residues for TECR and HACD2. Besides, we revealed the structural similarities of HACD with ELOVLs and proposed the possible competition mechanism of TECR-associated complex formation.

Fine-tuning activation specificity of G-protein-coupled receptors via automated path searching.

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.

Mechanism of agonist-induced activation of the human itch receptor MRGPRX1.

Mas-related G-protein-coupled receptors X1-X4 (MRGPRX1-X4) are 4 primate-specific receptors that are recently reported to be responsible for many biological processes, including itch sensation, pain transmission, and inflammatory reactions. MRGPRX1 is the first identified human MRGPR, and its expression is restricted to primary sensory neurons. Due to its dual roles in itch and pain signaling pathways, MRGPRX1 has been regarded as a promising target for itch remission and pain inhibition. Here, we reported a cryo-electron microscopy (cryo-EM) structure of Gq-coupled MRGPRX1 in complex with a synthetic agonist compound 16 in an active conformation at an overall resolution of 3.0 Å via a NanoBiT tethering strategy. Compound 16 is a new pain-relieving compound with high potency and selectivity to MRGPRX1 over other MRGPRXs and opioid receptor. MRGPRX1 was revealed to share common structural features of the Gq-mediated receptor activation mechanism of MRGPRX family members, but the variable residues in orthosteric pocket of MRGPRX1 exhibit the unique agonist recognition pattern, potentially facilitating to design MRGPRX1-specific modulators. Together with receptor activation and itch behavior evaluation assays, our study provides a structural snapshot to modify therapeutic molecules for itch relieving and analgesia targeting MRGPRX1.

An LC-MS-based workflow measures the export activity of S1P transporters.

Sphingosine-1-phosphate (S1P) is an active signaling metabolite synthesized by blood cells, exported into blood stream, and can trigger many downstream signaling pathways with disease implications. Understanding how S1P is transported is of great values for dissecting the function of S1P, but most existing methods for measuring S1P transporter activity use radioactive substrates or involve multiple workup steps, hindering their broader uses. In this study, we develop a workflow combining sensitive LC-MS measurement and a cell-based transporter protein system to measure the export activity of S1P transporter proteins. Our workflow demonstrated good applications in studying different S1P transporters SPNS2 and MFSD2B, WT and mutated protein, and different protein substrates. In summary, we provide a simple yet versatile workflow for measuring the export activity of S1P transporters, facilitating future studies of S1P transport mechanism and drug development.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

Exploring the Chemical Space of CYP17A1 Inhibitors Using Cheminformatics and Machine Learning.

Cytochrome P450 17A1 (CYP17A1) is one of the key enzymes in steroidogenesis that produces dehydroepiandrosterone (DHEA) from cholesterol. Abnormal DHEA production may lead to the progression of severe diseases, such as prostatic and breast cancers. Thus, CYP17A1 is a druggable target for anti-cancer molecule development. In this study, cheminformatic analyses and quantitative structure-activity relationship (QSAR) modeling were applied on a set of 962 CYP17A1 inhibitors (i.e., consisting of 279 steroidal and 683 nonsteroidal inhibitors) compiled from the ChEMBL database. For steroidal inhibitors, a QSAR classification model built using the PubChem fingerprint along with the extra trees algorithm achieved the best performance, reflected by the accuracy values of 0.933, 0.818, and 0.833 for the training, cross-validation, and test sets, respectively. For nonsteroidal inhibitors, a systematic cheminformatic analysis was applied for exploring the chemical space, Murcko scaffolds, and structure-activity relationships (SARs) for visualizing distributions, patterns, and representative scaffolds for drug discoveries. Furthermore, seven total QSAR classification models were established based on the nonsteroidal scaffolds, and two activity cliff (AC) generators were identified. The best performing model out of these seven was model VIII, which is built upon the PubChem fingerprint along with the random forest algorithm. It achieved a robust accuracy across the training set, the cross-validation set, and the test set, i.e., 0.96, 0.92, and 0.913, respectively. It is anticipated that the results presented herein would be instrumental for further CYP17A1 inhibitor drug discovery efforts.

Structural insights into sphingosine-1-phosphate receptor activation.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

Molecular mechanism of mitochondrial phosphatidate transfer by Ups1.

Cardiolipin, an essential mitochondrial physiological regulator, is synthesized from phosphatidic acid (PA) in the inner mitochondrial membrane (IMM). PA is synthesized in the endoplasmic reticulum and transferred to the IMM via the outer mitochondrial membrane (OMM) under mediation by the Ups1/Mdm35 protein family. Despite the availability of numerous crystal structures, the detailed mechanism underlying PA transfer between mitochondrial membranes remains unclear. Here, a model of Ups1/Mdm35-membrane interaction is established using combined crystallographic data, all-atom molecular dynamics simulations, extensive structural comparisons, and biophysical assays. The α2-loop, L2-loop, and α3 helix of Ups1 mediate membrane interactions. Moreover, non-complexed Ups1 on membranes is found to be a key transition state for PA transfer. The membrane-bound non-complexed Ups1/ membrane-bound Ups1 ratio, which can be regulated by environmental pH, is inversely correlated with the PA transfer activity of Ups1/Mdm35. These results demonstrate a new model of the fine conformational changes of Ups1/Mdm35 during PA transfer.

SmartBac, a new baculovirus system for large protein complex production.

Recent revolution of cryo-electron microscopy has opened a new door to solve high-resolution structures of macromolecule complexes without crystallization while how to efficiently obtain homogenous macromolecule complex sample is therefore becoming a bottleneck. Here we report SmartBac, an easy and versatile system for constructing large-sized transfer plasmids used to generate recombinant baculoviruses that express large multiprotein complexes in insect cells. The SmartBac system integrates the univector plasmid-fusion system, Gibson assembly method and polyprotein strategy to construct the final transfer plasmid. The fluorescent proteins are designed co-expressed with the target to monitor transfection and expression efficiencies. A scheme of screening an optimal tagged subunit for efficient purification is provided. Six large multiprotein complexes including the human exocyst complex and dynactin complex were successfully expressed and purified, suggesting a great potential of SmartBac system for its wide application in the future.