Ross T. Fernley

Structure of the insulin receptor ectodomain reveals a folded-over conformation

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 Å resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.

Inhibition studies of sulfonamide-containing folate analogs in yeast.

In the folate biosynthetic pathway, sulfa drugs (sulfonamides and sulfones) compete with the natural substrate, para-aminobenzoate (pABA) causing depletion of dihydrofolate (DHF) and subsequent growth inhibition. The sulfa drugs condense with 2-amino-4-hydroxy-6-hydroxymethyl-7,8 dihydropteridine pyrophosphate (DHPPP) forming sulfa-dihydropteroate (sulfa-DHP). Here evidence is presented using yeast that such dihydropteroate (DHP) analogs are inhibitory through competition with DHF. Two folate synthesis mutants, with respective dihydrofolate synthase (DHFS) and dihydropteroate synthase (DHPS) deletions and requiring DHF for growth were exposed to sulfa drugs. The DHFS knockout mutant was inhibited, but the DHPS knockout mutant that was incapable of forming sulfa-DHP was insensitive. Such sulfa-DHP compounds were chemically synthesized and shown to be inhibitory in vivo by competing with DHF, but in vitro assays with double the concentration of the sulfa-DHP to DHF showed no inhibition of dihydrofolate reductase (DHFR). Sequence analysis of resistant mutants obtained in the presence of sulfa drugs showed no changes in DHFR, or DHPS, unlike previously found antifolate-resistant mutants. The diamino derivatives, which are precursors of the sulfa-DHP, were found to be DHFR inhibitors. These results suggest that a new class of drugs, based on DHP analogs, could be investigated.

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor.

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, Kd, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and 15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including 15N-1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.

An ortholog of the ecdysone receptor protein (EcR) from the parasitic nematode Haemonchus contortus.

High concentrations (> or =4.2mM) of 20E inhibit the development of Haemonchus contortus eggs to the L3 larval stage. We report the cloning of cDNA encoding an EcR ortholog (HcEcR) from H. contortus mRNA expressed during L3. Phylogenetically, this and the putative EcR from Brugia malayi form a separate branch between arthropod EcRs and liver X receptors. Two isoforms of HcEcR differ in the inclusion/omission of a 3-residue segment in the A/B domain. Single nucleotide polymorphisms at 49 positions can be grouped into two major patterns in the A/BC segment and two in the DE/F segment. Some 35% of the highly conserved ecdysteroid-contacting residues in insect EcRs are also conserved in the HcEcR ligand binding domain, but it contains unusual residue choices at other ligand-contacting positions. Recombinant co-expression of HcEcR DE/F segments with a phthirapteran USP DE/F segment in insect cells resulted in stable proteins which did not heterodimerize or bind [(3)H]ponasterone A.

Chapter Four – Structure and Function of Ecdysone Receptors—Interactions with Ecdysteroids and Synthetic Agonists

Abstract The binding of ecdysteroids and the bisacylhydrazine insecticide, tebufenozide, to recombinant ecdysone receptor ligand-binding domains from pest insects points to conserved and variable features of the receptors ligand-binding pocket. Fluorophores conjugated to the terminus of the ecdysteroid alkyl chain have surprisingly little effect on receptor binding, permitting the development of a fluorescence polarization chemical library screen that has led to the discovery of a new class of ecdysone receptor ligands, the methylene lactams. X-ray structures of ecdysone receptor ligand-binding domains have allowed identification of the conserved and variable features within the binding pocket. The structures offer explanations for the lepidopteran selectivity of the bisacylhydrazines, the effect of amino acid replacements on the binding of ecdysteroids and other chemistries, and the preference of a phytophagous pentatomomorphan for makisterone A; indeed, they speak to the control spectra of future ecdysone receptor-targeting insecticides. Possible ligands for nematode ecdysone receptor orthologs are also considered.

Expression of HIV-1 nef in Yeast Causes Membrane Perturbation and Release of the Myristylated Nef Protein

The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.

Unprecedented conformational flexibility revealed in the ligand-binding domains of the Bovicola ovis ecdysone receptor (EcR) and ultraspiracle (USP) subunits.

The heterodimeric ligand-binding region of the Bovicola ovis ecdysone receptor has been crystallized either in the presence of an ecdysteroid or a synthetic methylene lactam insecticide. Two X-ray crystallographic structures, determined at 2.7 Å resolution, show that the ligand-binding domains of both subunits of this receptor, like those of other nuclear receptors, can display significant conformational flexibility. Thermal melt experiments show that while ponasterone A stabilizes the higher order structure of the heterodimer in solution, the methylene lactam destabilizes it. The conformations of the EcR and USP subunits observed in the structure crystallized in the presence of the methylene lactam have not been seen previously in any ecdysone receptor structure and represent a new level of conformational flexibility for these important receptors. Interestingly, the new USP conformation presents an open, unoccupied ligand-binding pocket.

Crystallization and preliminary X-ray analysis of 6-­hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Staphylococcus aureus

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 A, alpha = gamma = 90.0, beta = 100.2 degrees . The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V(M)) of 2.04 A(3) Da(-1) and an estimated solvent content of 39.6%.