Phote-HrTH (Phormia terraenovae Hypertrehalosaemic Hormone), the Metabolic Hormone of the Fruit Fly: Solution Structure and Receptor Binding Model

2020 ◽  
Vol 73 (3) ◽  
pp. 202
Author(s):  
Ibrahim A. Abdulganiyyu ◽  
Marc-Antoine Sani ◽  
Frances Separovic ◽  
Heather Marco ◽  
Graham E. Jackson
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Solution Structure ◽  
Sodium Dodecyl ◽  
Fruit Fly ◽  
Food Sources ◽  
Receptor Interaction ◽  
Binding Model ◽  
Model Based ◽  
Species Specific

Fruit flies are a widely distributed pest insect that pose a significant threat to food security. Flight is essential for the dispersal of the adult flies to find new food sources and ideal breeding spots. The supply of metabolic fuel to power the flight muscles of insects is regulated by adipokinetic hormones (AKHs). The fruit fly, Drosophila melanogaster, has the same AKH that is present in the blowfly, Phormia terraenovae; this AKH has the code-name Phote-HrTH. Binding of the AKH to the extra-cellular binding site of a G protein-coupled receptor causes its activation. In this paper, the structure of Phote-HrTH in sodium dodecyl sulfate (SDS) micelle solution was determined using NMR restrained molecular dynamics. The peptide was found to bind to the micelle and be fairly rigid, with an S2 order parameter of 0.96. The translated protein sequence of the AKH receptor from the fruit fly, D. melanogaster, Drome-AKHR, was used to construct two models of the receptor. It is proposed that these two models represent the active and inactive state of the receptor. The model based on the crystal structure of the β-2 adrenergic receptor was found to bind Phote-HrTH with a binding constant of −102kJmol−1, while the other model, based on the crystal structure of rhodopsin, did not bind the peptide. Under molecular dynamic simulation, in a palmitoyloleoylphosphatidylcholine (POPC) membrane, the receptor complex changed from an inactive to an active state. The identification and characterisation of the ligand binding site of Drome-AKHR provide novel information of ligand–receptor interaction, which could lead to the development of species-specific control substances to use discriminately against the fruit fly.

scholarly journals Heptad Repeat 2-Based Peptides Inhibit Avian Sarcoma and Leukosis Virus Subgroup A Infection and Identify a Fusion Intermediate

2004 ◽  
Vol 78 (24) ◽  
pp. 13430-13439 ◽  
Author(s):  
Robert C. Netter ◽  
Sean M. Amberg ◽  
John W. Balliet ◽  
Mark J. Biscone ◽  
Arwen Vermeulen ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heptad Repeat ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Envelope Conformation ◽  
Single Amino Acid Change ◽  
Avian Sarcoma

ABSTRACT Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the avian sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4°C became sensitive to peptide within the first 5 min of elevating the temperature to 37°C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.

scholarly journals Defining the LGR8 Residues Involved in Binding Insulin-Like Peptide 3

2007 ◽  
Vol 21 (7) ◽  
pp. 1699-1712 ◽  
Author(s):  
Daniel J. Scott ◽  
Tracey N. Wilkinson ◽  
Suode Zhang ◽  
Tania Ferraro ◽  
John D. Wade ◽  
...  
Keyword(s):  
Binding Site ◽  
Solution Structure ◽  
Peptide Hormone ◽  
Receptor Interaction ◽  
Testicular Descent ◽  
Binding Assays ◽  
Interaction Point ◽  
Multiple Sequence ◽  
Competition Binding ◽  
Leucine Rich Repeats

Abstract The peptide hormone insulin-like peptide 3 (INSL3) is essential for testicular descent and has been implicated in the control of adult fertility in both sexes. The human INSL3 receptor leucine-rich repeat-containing G protein-coupled receptor 8 (LGR8) binds INSL3 and relaxin with high affinity, whereas the relaxin receptor LGR7 only binds relaxin. LGR7 and LGR8 bind their ligands within the 10 leucine-rich repeats (LRRs) that comprise the majority of their ectodomains. To define the primary INSL3 binding site in LGR8, its LRRs were first modeled on the crystal structure of the Nogo receptor (NgR) and the most likely binding surface identified. Multiple sequence alignment of this surface revealed the presence of seven of the nine residues implicated in relaxin binding to LGR7. Replacement of these residues with alanine caused reduced [125I]INSL3 binding, and a specific peptide/receptor interaction point was revealed using competition binding assays with mutant INSL3 peptides. This point was used to crudely dock the solution structure of INSL3 onto the LRR model of LGR8, allowing the prediction of the INSL3 Trp-B27 binding site. This prediction was then validated using mutant INSL3 peptide competition binding assays on LGR8 mutants. Our results indicated that LGR8 Asp-227 was crucial for binding INSL3 Arg-B16, whereas LGR8 Phe-131 and Gln-133 were involved in INSL3 Trp-B27 binding. From these two defined interactions, we predicted the complete INSL3/LGR8 primary binding site, including interactions between INSL3 His-B12 and LGR8 Trp-177, INSL3 Val-B19 and LGR8 Ile-179, and INSL3 Arg-B20 with LGR8 Asp-181 and Glu-229.

scholarly journals The crystal structure of poplar apoplastocyanin at 1.8-A resolution. The geometry of the copper-binding site is created by the polypeptide.

1984 ◽  
Vol 259 (5) ◽  
pp. 2822-2825 ◽  
Author(s):  
T P Garrett ◽  
D J Clingeleffer ◽  
J M Guss ◽  
S J Rogers ◽  
H C Freeman
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Copper Binding

FLAVIN BINDING SITE GEOMETRY IN ANABAENA 7120 FLAVODOXIN. PROGRESS IN DETERMINING THE FLAVODOXIN SOLUTION STRUCTURE

1991 ◽  
pp. 377-380
Author(s):  
Brian J. Stockman ◽  
Andrzej M. Krezcl ◽  
John B. Olson ◽  
Ed S. Mooberry ◽  
John L. Markley
Keyword(s):  
Binding Site ◽  
Solution Structure ◽  
Anabaena 7120 ◽  
Flavin Binding

scholarly journals A Model-Based Volume Estimator that Accounts for Both Land Cover Misclassification and Model Prediction Uncertainty

2020 ◽  
Vol 12 (20) ◽  
pp. 3360
Author(s):  
Jessica Esteban ◽  
Ronald E. McRoberts ◽  
Alfredo Fernández-Landa ◽  
José Luis Tomé ◽  
Miguel Marchamalo
Keyword(s):  
Forest Inventory ◽  
Laser Scanning ◽  
Specific Area ◽  
Training Data ◽  
Forest Species ◽  
Total Uncertainty ◽  
Estimation Process ◽  
Model Based ◽  
Species Specific ◽  
Field Plots

Forest/non-forest and forest species maps are often used by forest inventory programs in the forest estimation process. For example, some inventory programs establish field plots only on lands corresponding to the forest portion of a forest/non-forest map and use species-specific area estimates obtained from those maps to support the estimation of species-specific volume (V) totals. Despite the general use of these maps, the effects of their uncertainties are commonly ignored with the result that estimates might be unreliable. The goal of this study is to estimate the effects of the uncertainty of forest species maps used in the sampling and estimation processes. Random forest (RF) per-pixel predictions were used with model-based inference to estimate V per unit area for the six main forest species of La Rioja, Spain. RF models for predicting V were constructed using field plot information from the Spanish National Forest Inventory and airborne laser scanning data. To limit the prediction of V to pixels classified as one of the main forest species assessed, a forest species map was constructed using Landsat and auxiliary information. Bootstrapping techniques were implemented to estimate the total uncertainty of the V estimates and accommodated both the effects of uncertainty in the Landsat forest species map and the effects of plot-to-plot sampling variability on training data used to construct the RF V models. Standard errors of species-specific total V estimates increased from 2–9% to 3–22% when the effects of map uncertainty were incorporated into the uncertainty assessment. The workflow achieved satisfactory results and revealed that the effects of map uncertainty are not negligible, especially for open-grown and less frequently occurring forest species for which greater variability was evident in the mapping and estimation process. The effects of forest map uncertainty are greater for species-specific area estimation than for the selection of field plots used to calibrate the RF model. Additional research to generalize the conclusions beyond Mediterranean to other forest environments is recommended.

scholarly journals Crystal structure of thermostable alkylsulfatase SdsAP from Pseudomonas sp. S9

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Lifang Sun ◽  
Pu Chen ◽  
Yintao Su ◽  
Zhixiong Cai ◽  
Lingwei Ruan ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sodium Dodecyl ◽  
Titration Calorimetry ◽  
Pseudomonas Sp ◽  
Sterol Carrier Protein ◽  
Dimerization Domain ◽  
Alkyl Chains ◽  
Hydrolysis Of

A novel alkylsulfatase from bacterium Pseudomonas sp. S9 (SdsAP) was identified as a thermostable alkylsulfatases (type III), which could hydrolyze the primary alkyl sulfate such as sodium dodecyl sulfate (SDS). Thus, it has a potential application of SDS biodegradation. The crystal structure of SdsAP has been solved to a resolution of 1.76 Å and reveals that SdsAP contains the characteristic metallo-β-lactamase-like fold domain, dimerization domain, and C-terminal sterol carrier protein type 2 (SCP-2)-like fold domain. Kinetic characterization of SdsAP to SDS by isothermal titration calorimetry (ITC) and enzymatic activity assays of constructed mutants demonstrate that Y246 and G263 are important residues for its preference for the hydrolysis of ‘primary alkyl’ chains, confirming that SdsAP is a primary alkylsulfatase.

scholarly journals Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase

2005 ◽  
Vol 187 (7) ◽  
pp. 2386-2394 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Andrea Gorrell ◽  
Sarah H. Lawrence ◽  
Prabha Iyer ◽  
Kerry Smith ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Binding Pocket ◽  
Acetate Kinase ◽  
Phosphoryl Group ◽  
Acetyl Phosphate ◽  
Hydrophobic Pocket ◽  
Methanosarcina Thermophila ◽  
Methyl Group ◽  
Substrate Binding Sites

ABSTRACT Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val93, Leu122, Phe179, and Pro232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with Km values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

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