Karsten Melcher

Van Andel Research Institute, Grand Rapids, Michigan, United States

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Publications (42)481.35 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2-3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.
    Acta pharmacologica Sinica. 06/2014;
  • Ley Moy Ng, Karsten Melcher, Bin Tean Teh, H Eric Xu
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    ABSTRACT: Adverse environmental conditions are a threat to agricultural yield and therefore exert a global effect on livelihood, health and the economy. Abscisic acid (ABA) is a vital plant hormone that regulates abiotic stress tolerance, thereby allowing plants to cope with environmental stresses. Previously, attempts to develop a complete understanding of the mechanisms underlying ABA signaling have been hindered by difficulties in the identification of bona fide ABA receptors. The discovery of the PYR/PYL/RCAR family of ABA receptors therefore represented a major milestone in the effort to overcome these roadblocks; since then, many structural and functional studies have provided detailed insights into processes ranging from ABA perception to the activation of ABA-responsive gene transcription. This understanding of the mechanisms of ABA perception and signaling has served as the basis for recent, preliminary developments in the genetic engineering of stress-resistant crops as well as in the design of new synthetic ABA agonists, which hold great promise for the agricultural enhancement of stress tolerance.
    Acta Pharmacologica Sinica 05/2014; 35(5):567-584. · 2.35 Impact Factor
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    ABSTRACT: 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.Cell Research advance online publication 25 April 2014; doi:10.1038/cr.2014.52.
    Cell Research 04/2014; · 10.53 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
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    ABSTRACT: Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCF(D3) ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCF(D3) ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.
    Nature 12/2013; · 38.60 Impact Factor
  • Kuntal Pal, Karsten Melcher, H Eric Xu
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    ABSTRACT: Class B G-protein-coupled receptors are exciting drug targets, yet the structure of a complete receptor bound to a peptide agonist has remained elusive. Coin et al. present a model of the receptor CRF1R bound to its native ligand based on partial structures and 44 spatial constraints revealed by new crosslinking approaches.
    Cell 12/2013; 155(6):1207-8. · 31.96 Impact Factor
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    ABSTRACT: Thylakoid assembly 8 (THA8) is a pentatricopeptide repeat (PPR) RNA-binding protein required for the splicing of the transcript of ycf3, a gene involved in chloroplast thylakoid-membrane biogenesis. Here we report the identification of multiple THA8-binding sites in the ycf3 intron and present crystal structures of Brachypodium distachyon THA8 either free of RNA or bound to two of the identified RNA sites. The apostructure reveals a THA8 monomer with five tandem PPR repeats arranged in a planar fold. The complexes of THA8 bound to the two short RNA fragments surprisingly reveal asymmetric THA8 dimers with the bound RNAs at the dimeric interface. RNA binding induces THA8 dimerization, with a conserved G nucleotide of the bound RNAs making extensive contacts with both monomers. Together, these results establish a new model of RNA recognition by RNA-induced formation of an asymmetric dimer of a PPR protein.
    Nature Structural & Molecular Biology 11/2013; · 11.90 Impact Factor
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    ABSTRACT: Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.
    Genes & development 11/2013; 27(21):2305-19. · 12.08 Impact Factor
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    ABSTRACT: Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA binding proteins, which form a pervasive family of proteins conserved in yeast, plants, and humans. The plant PPR proteins are mainly grouped into P and PLS classes. Here we report the crystal structure of a PLS-class PPR protein from Arabidopsis thaliana, termed THA8-like (THA8L) at 2.0 Å. THA8L resembles the thylakoid assembly 8 (THA8), a protein that is required for the splicing of specific group II introns of genes involved in biogenesis of chloroplast thylakoid membranes. The THA8L structure contains three P-type PPR motifs flanked by one L-type motif and one S-type motif. We identified several putative THA8L-binding sites, enriched with purine sequences, in the group II introns. Importantly, THA8L has strong preferential binding for single stranded RNA over single stranded DNA or double stranded RNA. Structural analysis reveals that THA8L contains two extensive patches of positively charged residues next to the residues that are proposed to comprise the RNA-binding codes. Mutations in these two positively charged patches greatly reduced THA8L RNA-binding activity. Based on these data, we constructed a model of THA8L-RNA binding that is dependent on two forces: one is the interaction between nucleotide bases and specific amino acids in the PPR motifs (codes) and the other is the interaction between negatively charged RNA backbone and positively charged residues of PPR motifs. Together, these results further our understanding of the mechanism of PPR protein-RNA interactions.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: Folate receptors (FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate cellular uptake of folate. Although expressed at very low levels in most tissues, folate receptors, especially FRα, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions. The folate dependency of many tumours has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, high-affinity antifolates, folate-based imaging agents and folate-conjugated drugs and toxins. To understand how folate binds its receptors, we determined the crystal structure of human FRα in complex with folic acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulphide bonds and contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for designing more specific drugs targeting the folate receptor system.
    Nature 07/2013; · 38.60 Impact Factor
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    ABSTRACT: Abscisic acid (ABA) is the most important hormone for plants to resist drought and other abiotic stresses. ABA binds directly to the PYR/PYL family of ABA receptors, resulting in inhibition of type 2C phosphatases (PP2C) and activation of downstream ABA signaling. It is envisioned that intervention of ABA signaling by small molecules could help plants to overcome abiotic stresses such as drought, cold and soil salinity. However, chemical instability and rapid catabolism by plant enzymes limit the practical application of ABA itself. Here we report the identification of a small molecule ABA mimic (AM1) that acts as a potent activator of multiple members of the family of ABA receptors. In Arabidopsis, AM1 activates a gene network that is highly similar to that induced by ABA. Treatments with AM1 inhibit seed germination, prevent leaf water loss, and promote drought resistance. We solved the crystal structure of AM1 in complex with the PYL2 ABA receptor and the HAB1 PP2C, which revealed that AM1 mediates a gate-latch-lock interacting network, a structural feature that is conserved in the ABA-bound receptor/PP2C complex. Together, these results demonstrate that a single small molecule ABA mimic can activate multiple ABA receptors and protect plants from water loss and drought stress. Moreover, the AM1 complex crystal structure provides a structural basis for designing the next generation of ABA-mimicking small molecules.Cell Research advance online publication 9 July 2013; doi:10.1038/cr.2013.95.
    Cell Research 07/2013; · 10.53 Impact Factor
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    ABSTRACT: Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the accompanying structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3-angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.
    Science 03/2013; · 31.20 Impact Factor
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    Cell Research 02/2013; · 10.53 Impact Factor
  • Ley-Moy Ng, Fen-Fen Soon, Karsten Melcher
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    ABSTRACT: Graphical Abstract Figure optionsView in workspace Highlights ► Conformations support PYL2 “gate” role in dimerization and phosphatase inhibition ► Structural mechanism of ABA-PYL2 affinity for HAB1 over kinases linked to Trp lock ► HDX suggests kinase activity is associated with a stable “closed” conformation
    Structure 02/2013; 21(2):229–235. · 5.99 Impact Factor
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    ABSTRACT: Photoreceptor-specific nuclear receptor (PNR, NR2E3) is a key transcriptional regulator of human photoreceptor differentiation and maintenance. Mutations in the NR2E3-encoding gene cause various retinal degenerations, including Enhanced S-cone syndrome, retinitis pigmentosa, and Goldman-Favre disease. Although physiological ligands have not been identified, it is believed that binding of small molecule agonists, receptor desumoylation, and receptor heterodimerization may switch NR2E3 from a transcriptional repressor to an activator. While these features make NR2E3 a potential therapeutic target for the treatment of retinal diseases, there has been a clear lack of structural information for the receptor. Here, we report the crystal structure of the apo NR2E3 ligand binding domain (LBD) at 2.8 Å resolution. Apo NR2E3 functions as transcriptional repressor in cells and the structure of its LBD is in a dimeric auto-repressed conformation. In this conformation, the putative ligand binding pocket is filled with bulky hydrophobic residues and the activation-function-2 (AF2) helix occupies the canonical cofactor binding site. Mutations designed to disrupt either the AF2/cofactor-binding site interface or the dimer interface compromised the transcriptional repressor activity of this receptor. Together, these results reveal several conserved structural features shared by related orphan nuclear receptors, suggest that most disease-causing mutations affect the receptor's structural integrity, and allowed us to model a putative active conformation that can accommodate small ligands in its pocket.
    PLoS ONE 01/2013; 8(9):e74359. · 3.53 Impact Factor
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    ABSTRACT: Plants regulate growth and respond to environmental stress through abscisic acid (ABA) regulated pathways, and as such these pathways are of primary interest for biological and agricultural research. The ABA response is first perceived by the PYR/PYL/RCAR class of START protein receptors. These ABA activated receptors disrupt phosphatase inhibition of Snf1-related kinases (SnRKs), enabling kinase signaling. Here, insights into the structural mechanism of proteins in the ABA signaling pathway (the ABA receptor PYL2, HAB1 phosphatase, and two kinases, SnRK2.3 and 2.6) are discerned through hydrogen/deuterium exchange (HDX) mass spectrometry. HDX on the phosphatase in the presence of binding partners provides evidence for receptor-specific conformations involving the Trp385 "lock" that is necessary for signaling. Furthermore, kinase activity is linked to a more stable "closed" conformation. These solution-based studies complement the static crystal structures and provide a more detailed understanding of the ABA signaling pathway.
    Structure 12/2012; · 5.99 Impact Factor
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    ABSTRACT: 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.
    Plant signaling & behavior 05/2012; 7(5):581-8.
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    X Edward Zhou, Karsten Melcher, H Eric Xu
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    ABSTRACT: Rhodopsin is the first G-protein-coupled receptor (GPCR) with its three-dimensional structure solved by X-ray crystallography. The crystal structure of rhodopsin has revealed the molecular mechanism of photoreception and signal transduction in the visual system. Although several other GPCR crystal structures have been reported over the past few years, the rhodopsin structure remains an important model for understanding the structural and functional characteristics of other GPCRs. This review summarizes the structural features, the photoactivation, and the G protein signal transduction of rhodopsin.
    Acta Pharmacologica Sinica 03/2012; 33(3):291-9. · 2.35 Impact Factor
  • Kuntal Pal, Karsten Melcher, H Eric Xu
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    ABSTRACT: Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon, parathyroid hormone, and calcitonin. These receptors are involved in a wide spectrum of physiological activities, from metabolic regulation and stress control to development and maintenance of the skeletal system. As such, they are important drug targets for the treatment of diabetes, osteoporosis, and stress related disorders. Class B GPCRs are organized into two modular domains: an extracellular domain (ECD) and a helical bundle that contains seven transmembrane helices (TM domain). The ECD is responsible for the high affinity and specificity of hormone binding, and the TM domain is required for receptor activation and signal coupling to downstream G-proteins. Although the structure of the full-length receptor remains unknown, the ECD structures have been well characterized for a number of Class B GPCRs, revealing a common fold for ligand recognition. This review summarizes the general structural principles that guide hormone binding by Class B ECDs and their implications in the design of peptide hormone analogs for therapeutic purposes.
    Acta Pharmacologica Sinica 03/2012; 33(3):300-11. · 2.35 Impact Factor
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    ABSTRACT: 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.
    PLoS ONE 01/2012; 7(10):e47857. · 3.53 Impact Factor

Publication Stats

973 Citations
481.35 Total Impact Points

Institutions

  • 2009–2014
    • Van Andel Research Institute
      Grand Rapids, Michigan, United States
  • 2013
    • Chinese Academy of Sciences
      Peping, Beijing, China
    • The Scripps Research Institute
      La Jolla, California, United States
  • 2005–2010
    • University of Ulster
      • School of Biomedical Sciences
      Belfast, NIR, United Kingdom
  • 1992–2005
    • Goethe-Universität Frankfurt am Main
      Frankfurt, Hesse, Germany
  • 1998–2001
    • University of Texas Southwestern Medical Center
      • Department of Internal Medicine
      Dallas, TX, United States