Assembly of JAZ-JAZ and JAZ-NINJA complexes in jasmonate signaling.

Jasmonates (JAs) are plant hormones with crucial roles in development and stress resilience. They activate MYC transcription factors by mediating the proteolysis of MYC inhibitors called JAZ proteins. In the absence of JA, JAZ proteins bind and inhibit MYC through the assembly of MYC-JAZ-Novel Interactor of JAZ (NINJA)-TPL repressor complexes. However, JAZ and NINJA are predicted to be largely intrinsically unstructured, which has precluded their experimental structure determination. Through a combination of biochemical, mutational, and biophysical analyses and AlphaFold-derived ColabFold modeling, we characterized JAZ-JAZ and JAZ-NINJA interactions and generated models with detailed, high-confidence domain interfaces. We demonstrate that JAZ, NINJA, and MYC interface domains are dynamic in isolation and become stabilized in a stepwise order upon complex assembly. By contrast, most JAZ and NINJA regions outside of the interfaces remain highly dynamic and cannot be modeled in a single conformation. Our data indicate that the small JAZ Zinc finger expressed in Inflorescence Meristem (ZIM) motif mediates JAZ-JAZ and JAZ-NINJA interactions through separate surfaces, and our data further suggest that NINJA modulates JAZ dimerization. This study advances our understanding of JA signaling by providing insights into the dynamics, interactions, and structure of the JAZ-NINJA core of the JA repressor complex.

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Šresolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

Identification and structural insight of an effective PPARγ modulator with improved therapeutic index for anti-diabetic drug discovery.

Peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of glucose homeostasis and lipid metabolism, and an important target for the development of modern anti-diabetic drugs. However, current PPARγ-targeting anti-diabetic drugs such as classical thiazolidinediones (TZDs) are associated with undesirable side effects. To address this concern, we here describe the structure-based design, synthesis, identification and detailed in vitro and in vivo characterization of a novel, decanoic acid (DA)-based and selective PPARγ modulator (SPPARγM), VSP-77, especially (S)-VSP-77, as the potential "hit" for the development of improved and safer anti-diabetic therapeutics. We have also determined the co-crystal structure of the PPARγ ligand-binding domain (LBD) in complex with two molecules of (S)-VSP-77, which reveal a previously undisclosed allosteric binding mode. Overall, these findings not only demonstrate the therapeutic advantage of (S)-VSP-77 over current TZD drugs and representative partial agonist INT131, but also provide a rational basis for the development of future SPPARγMs as safe and highly efficacious anti-diabetic drugs.

Conformational heterogeneity of the allosteric drug and metabolite (ADaM) site in AMP-activated protein kinase (AMPK).

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and a promising drug target for managing metabolic diseases such as type 2 diabetes. Many pharmacological AMPK activators, and possibly unidentified physiological metabolites, bind to the allosteric drug and metabolite (ADaM) site at the interface between the kinase domain (KD) in the α-subunit and the carbohydrate-binding module (CBM) in the ß-subunit. Here, using double electron-electron resonance (DEER) spectroscopy, we demonstrate that the CBM-KD interaction is partially dissociated and the interface highly disordered in the absence of pharmacological ADaM site activators as inferred from a low depth of modulation and broad DEER distance distributions. ADaM site ligands such as 991, and to a lesser degree phosphorylation, stabilize the KD-CBM association and strikingly reduce conformational heterogeneity in the ADaM site. Our findings that the ADaM site, formed by the KD-CBM interaction, can be modulated by diverse ligands and by phosphorylation suggest that it may function as a hub for integrating regulatory signals.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

Identification of a novel selective PPARγ ligand with a unique binding mode and improved therapeutic profile in vitro.

Thiazolidinediones (TZD) function as potent anti-diabetic drugs through their direct action on the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), but their therapeutic benefits are compromised by severe side effects. To address this concern, here we developed a potent "hit" compound, VSP-51, which is a novel selective PPARγ-modulating ligand with improved therapeutic profiles in vitro compared to the multi-billion dollar TZD drug rosiglitazone (Rosi). Unlike Rosi, VSP-51 is a partial agonist of PPARγ with improved insulin sensitivity due to its ability to bind PPARγ with high affinity without stimulating adipocyte differentiation and the expression of adipogenesis-related genes. We have determined the crystal structure of the PPARγ ligand-binding domain (LBD) in complex with VSP-51, which revealed a unique mode of binding for VSP-51 and provides the molecular basis for the discrimination between VSP-51 from TZDs and other ligands such as telmisartan, SR1663 and SR1664. Taken together, our findings demonstrate that: a) VSP-51 can serve as a promising candidate for anti-diabetic drug discovery; and b) provide a rational basis for the development of future pharmacological agents targeting PPARγ with advantages over current TZD drugs.

X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex.

Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.

FXR Primes the Liver for Intestinal FGF15 Signaling by Transient Induction of ß-Klotho.

The bile acid (BA)-sensing nuclear receptor, farnesoid X receptor (FXR), regulates postprandial metabolic responses, including inhibition of BA synthesis, by inducing the intestinal hormone, fibroblast growth factor (FGF)15 (FGF19 in human). In this study, we tested a novel hypothesis that FXR not only induces intestinal FGF15 but also primes the liver for effectively responding to the signal by transcriptional induction of the obligate coreceptor for FGF15, ß-Klotho (ßKL). Activation of FXR by a synthetic agonist, GW4064, in mice increased occupancy of FXR and its DNA-binding partner, retinoid X receptor-α, at FGF15-signaling component genes, particularly ßKL, and induced expression of these genes. Interestingly, mRNA levels of Fgfr4, the FGF15 receptor, were not increased by GW4064, but protein levels increased as a result of ßKL-dependent increased protein stability. Both FGF receptor 4 and ßKL protein levels were substantially decreased in FXR-knockout (KO) mice, and FGF19 signaling, monitored by phosphorylated ERK, was blunted in FXR-KO mice, FXR-KO mouse hepatocytes, and FXR-down-regulated human hepatocytes. Overexpression of ßKL in FXR-lacking hepatocytes partially restored FGF19 signaling and inhibition by FGF19 of Cyp7a1, which encodes the rate-limiting BA biosynthetic enzyme. In mice, transient inductions of intestinal Fgf15 and hepatic ßKL were temporally correlated after GW4064 treatment, and pretreatment of hepatocytes with GW4064 before FGF19 treatment enhanced FGF19 signaling, which was abolished by transcriptional inhibition or ßKL down-regulation. This study identifies FXR as a gut-liver metabolic coordinator for FGF15/19 action that orchestrates transient induction of hepatic ßKL and intestinal Fgf15/19 in a temporally correlated manner.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.

MicroRNA 34a inhibits beige and brown fat formation in obesity in part by suppressing adipocyte fibroblast growth factor 21 signaling and SIRT1 function.

Brown fat generates heat through uncoupled respiration, protecting against hypothermia and obesity. Adult humans have brown fat, but the amounts and activities are substantially decreased in obesity, by unknown mechanisms. Here we show that elevated microRNA 34a (miR-34a) in obesity inhibits fat browning in part by suppressing the browning activators fibroblast growth factor 21 (FGF21) and SIRT1. Lentivirus-mediated downregulation of miR-34a in mice with diet-induced obesity reduced adiposity, improved serum profiles, increased the mitochondrial DNA copy number, and increased oxidative function in adipose tissue in both BALB/c and C57BL/6 mice. Remarkably, downregulation of miR-34a increased coexpression of the beige fat-specific marker CD137 and the browning marker UCP1 in all types of white fat, including visceral fat, and promoted additional browning in brown fat. Mechanistically, downregulation of miR-34a increased expression of the FGF21 receptor components, FGFR1 and ßKL, and also that of SIRT1, resulting in FGF21/SIRT1-dependent deacetylation of PGC-1α and induction of the browning genes Ucp1, Pgc-1α, and Prdm16. Importantly, anti-miR-34a-mediated beneficial effects, including decreased adiposity, are likely from multiple tissues, since downregulation of miR-34a also improves hepatic FGF21 signaling and lipid oxidation. This study identifies miR-34a as an inhibitor of beige and brown fat formation, providing a potential target for treating obesity-related diseases.

Cytoplasmic tyrosine phosphatase Shp2 coordinates hepatic regulation of bile acid and FGF15/19 signaling to repress bile acid synthesis.

Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR) and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that nonreceptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through the orchestration of multiple signals in hepatocytes.

Structures and mechanism for the design of highly potent glucocorticoids.

The evolution of glucocorticoid drugs was driven by the demand of lowering the unwanted side effects, while keeping the beneficial anti-inflammatory effects. Potency is an important aspect of this evolution as many undesirable side effects are associated with use of high-dose glucocorticoids. The side effects can be minimized by highly potent glucocorticoids that achieve the same treatment effects at lower doses. This demand propelled the continuous development of synthetic glucocorticoids with increased potencies, but the structural basis of their potencies is poorly understood. To determine the mechanisms underlying potency, we solved the X-ray structures of the glucocorticoid receptor (GR) ligand-binding domain (LBD) bound to its endogenous ligand, cortisol, which has relatively low potency, and a highly potent synthetic glucocorticoid, mometasone furoate (MF). The cortisol-bound GR LBD revealed that the flexibility of the C1-C2 single bond in the steroid A ring is primarily responsible for the low affinity of cortisol to GR. In contrast, we demonstrate that the very high potency of MF is achieved by its C-17α furoate group completely filling the ligand-binding pocket, thus providing additional anchor contacts for high-affinity binding. A single amino acid in the ligand-binding pocket, Q642, plays a discriminating role in ligand potency between MF and cortisol. Structure-based design led to synthesis of several novel glucocorticoids with much improved potency and efficacy. Together, these results reveal key structural mechanisms of glucocorticoid potency and provide a rational basis for developing novel highly potent glucocorticoids.

Structural insights into gene repression by the orphan nuclear receptor SHP.

Small heterodimer partner (SHP) is an orphan nuclear receptor that functions as a transcriptional repressor to regulate bile acid and cholesterol homeostasis. Although the precise mechanism whereby SHP represses transcription is not known, E1A-like inhibitor of differentiation (EID1) was isolated as a SHP-interacting protein and implicated in SHP repression. Here we present the crystal structure of SHP in complex with EID1, which reveals an unexpected EID1-binding site on SHP. Unlike the classical cofactor-binding site near the C-terminal helix H12, the EID1-binding site is located at the N terminus of the receptor, where EID1 mimics helix H1 of the nuclear receptor ligand-binding domain. The residues composing the SHP-EID1 interface are highly conserved. Their mutation diminishes SHP-EID1 interactions and affects SHP repressor activity. Together, these results provide important structural insights into SHP cofactor recruitment and repressor function and reveal a conserved protein interface that is likely to have broad implications for transcriptional repression by orphan nuclear receptors.

Bile acid signal-induced phosphorylation of small heterodimer partner by protein kinase Cζ is critical for epigenomic regulation of liver metabolic genes.

Bile acids (BAs) are recently recognized key signaling molecules that control integrative metabolism and energy expenditure. BAs activate multiple signaling pathways, including those of nuclear receptors, primarily farnesoid X receptor (FXR), membrane BA receptors, and FXR-induced FGF19 to regulate the fed-state metabolism. Small heterodimer partner (SHP) has been implicated as a key mediator of these BA signaling pathways by recruitment of chromatin modifying proteins, but the key question of how SHP transduces BA signaling into repressive histone modifications at liver metabolic genes remains unknown. Here we show that protein kinase Cζ (PKCζ) is activated by BA or FGF19 and phosphorylates SHP at Thr-55 and that Thr-55 phosphorylation is critical for the epigenomic coordinator functions of SHP. PKCζ is coimmunopreciptitated with SHP and both are recruited to SHP target genes after bile acid or FGF19 treatment. Activated phosphorylated PKCζ and phosphorylated SHP are predominantly located in the nucleus after FGF19 treatment. Phosphorylation at Thr-55 is required for subsequent methylation at Arg-57, a naturally occurring mutation site in metabolic syndrome patients. Thr-55 phosphorylation increases interaction of SHP with chromatin modifiers and their occupancy at selective BA-responsive genes. This molecular cascade leads to repressive modifications of histones at metabolic target genes, and consequently, decreased BA pools and hepatic triglyceride levels. Remarkably, mutation of Thr-55 attenuates these SHP-mediated epigenomic and metabolic effects. This study identifies PKCζ as a novel key upstream regulator of BA-regulated SHP function, revealing the role of Thr-55 phosphorylation in epigenomic regulation of liver metabolism.

Abscisic acid signaling: thermal stability shift assays as tool to analyze hormone perception and signal transduction.

Abscisic acid (ABA) is a plant hormone that plays important roles in growth and development. ABA is also the central regulator to protect plants against abiotic stresses, such as drought, high salinity, and adverse temperatures, and ABA signaling is therefore a promising biotechnological target for the generation of crops with increased stress resistance. Recently, a core signal transduction pathway has been established, in which ABA receptors, type 2C protein phosphatases, and AMPK-related protein kinases control the regulation of transcription factors, ion channels, and enzymes. Here we use a simple protein thermal stability shift assay to independently validate key aspects of this pathway and to demonstrate the usefulness of this technique to detect and characterize very weak (Kd ≥ 50 µM) interactions between receptors and physiological and synthetic agonists, to determine and analyze protein-protein interactions, and to screen small molecule inhibitors.

Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor ß-Klotho.

MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor ß-Klotho (ßKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired ßKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates ßKL by binding to the 3' UTR of ßKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic ßKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, ßKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored ßKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti-miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a ßKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting ßKL. The miR-34a/ßKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.

Catalytic mechanism and kinase interactions of ABA-signaling PP2C phosphatases.

Abscisic acid (ABA) is an essential hormone that controls plant growth, development and responses to abiotic stresses. ABA signaling is mediated by type 2C protein phosphatases (PP2Cs), including HAB1 and ABI2, which inhibit stress-activated SnRK2 kinases and whose activity is regulated by ABA and ABA receptors. Based on biochemical data and our previously determined crystal structures of ABI2 and the SnRK2.6-HAB1 complex, we present the catalytic mechanism of PP2C and provide new insight into PP2C-SnRK2 interactions and possible roles of other SnRK2 kinases in ABA signaling.

Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases.

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.