First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

ABSTRACT Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (Pck Tk ). Pck Tk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in Pck Tk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant Pck Tk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of Pck Tk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of Pck Tk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.

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Further Characterization of the Glucocorticoid Response Unit in the Phosphoenolpyruvate Carboxykinase Gene. The Role of the Glucocorticoid Receptor-Binding Sites

Molecular Endocrinology ◽

10.1210/mend.12.4.0090 ◽

1998 ◽

Vol 12 (4) ◽

pp. 482-491 ◽

Cited By ~ 28

Author(s):

Donald K. Scott ◽

Per-Erik Strömstedt ◽

Jen-Chywan Wang ◽

Daryl K. Granner

Keyword(s):

Glucocorticoid Receptor ◽

Receptor Binding ◽

Binding Sites ◽

Phosphoenolpyruvate Carboxykinase ◽

Response Unit ◽

Glucocorticoid Response ◽

Phosphoenolpyruvate Carboxykinase Gene

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Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

Blood ◽

10.1182/blood.v128.22.5056.5056 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5056-5056 ◽

Cited By ~ 2

Author(s):

Julia Fremerey ◽

Pavel Morozov ◽

Cindy Meyer ◽

Aitor Garzia ◽

Marianna Teplova ◽

...

Keyword(s):

Mass Spectrometry ◽

Binding Sites ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Binding Properties ◽

Expression Levels ◽

Rna Targets ◽

Polymerase I

Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

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Insight into the essential role of the Helicobacter pylori HP1043 orphan response regulator: genome-wide identification and characterization of the DNA-binding sites

Scientific Reports ◽

10.1038/srep41063 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 14

Author(s):

Simone Pelliciari ◽

Eva Pinatel ◽

Andrea Vannini ◽

Clelia Peano ◽

Simone Puccio ◽

...

Keyword(s):

Helicobacter Pylori ◽

Binding Sites ◽

Essential Role ◽

Response Regulator ◽

Dna Binding Sites ◽

Genome Wide ◽

Identification And Characterization ◽

Insight Into

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The role of monovalent cations in the ATPase reaction of DNA gyrase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715002916 ◽

2015 ◽

Vol 71 (4) ◽

pp. 996-1005 ◽

Cited By ~ 8

Author(s):

Stephen James Hearnshaw ◽

Terence Tsz-Hong Chung ◽

Clare Elizabeth Mary Stevenson ◽

Anthony Maxwell ◽

David Mark Lawson

Keyword(s):

Escherichia Coli ◽

Binding Sites ◽

Dna Gyrase ◽

Monovalent Cation ◽

Cation Binding ◽

Monovalent Cations ◽

Atpase Domain ◽

Atpase Reaction ◽

Resolution Structure

Four new crystal structures of the ATPase domain of the GyrB subunit ofEscherichia coliDNA gyrase have been determined. One of these, solved in the presence of K+, is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K+ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na+ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.

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Role of structural water for prediction of cation binding sites in apoproteins

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2016.1273136 ◽

2017 ◽

Vol 36 (1) ◽

pp. 221-232 ◽

Cited By ~ 2

Author(s):

L.A. Uroshlev ◽

I.V. Kulakovskiy ◽

N.G. Esipova ◽

V.G. Tumanyan ◽

S.V. Rahmanov ◽

...

Keyword(s):

Binding Sites ◽

Cation Binding ◽

Structural Water

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Design of novel CV-N variants: Modulation of binding dynamics through distal mutations

10.1101/2020.12.21.423842 ◽

2020 ◽

Author(s):

I. Can Kazan ◽

Prerna Sharma ◽

Andrey Bobkov ◽

Raimund Fromme ◽

Giovanna Ghirlanda ◽

...

Keyword(s):

Binding Affinity ◽

Binding Sites ◽

Computational Method ◽

Experimental Characterization ◽

Dynamic Coupling ◽

Binding Region ◽

Novel Approach ◽

Allosteric Sites ◽

Docking Tool

AbstractWe develop a computational approach to identify distal residues that allosterically modulate the dynamics of binding sites by combining dynamic coupling with statistical analysis of co-evolution. Putative mutants of these predicted allosteric sites are subjected to Adaptive BP-Dock docking tool for binding analysis. Here, we apply this method to a small lectin, Cyanovirin-N (CV-N), that selectively binds to dimannose. Our computational method points out mutations on I34, that is 16Å away from binding site can modulate binding. Experimental characterization of I34 mutants confirms that I34Y increases affinity towards dimannose, while I34K completely abolish binding. The increased affinity is not due to changes in the binding region, which are conserved in the crystal structure. However, ITC analysis reveals an opposite contribution of TΔS (negative in WT, and positive in I34Y) and suggests that modulation of dynamics (i.e., dynamic allostery) is responsible for the change in binding affinity. Our results point to a novel approach to identify and substitute distal sites, guiding the mutational landscape in glycan-binding proteins to improve binding affinity.

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Demonstration and characterization of motilin-binding sites in the rabbit cerebellum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.272.5.g994 ◽

1997 ◽

Vol 272 (5) ◽

pp. G994-G999 ◽

Cited By ~ 6

Author(s):

I. Depoortere ◽

T. L. Peeters

Keyword(s):

Binding Sites ◽

Molecular Layer ◽

Physiological Role ◽

Scatchard Analysis ◽

Affinity Binding ◽

Lower Affinity ◽

Erythromycin A ◽

G Protein Coupled

This is the first report on central motilin receptors. Autoradiography on cerebellar slices revealed specific motilin-binding sites in the molecular layer of the cortex. Scatchard analysis of cold saturation studies showed the existence of a high-(pKd,hi = 9.07 +/- 0.09, where pKd is the negative logarithm of the dissociation constant) and a low-affinity binding site (pKd,lo = 6.56 +/- 0.09). Similar affinities were found with rabbit motilin and with the porcine (po) antagonist [Phe3, Leu13]po-motilin. Feline and canine motilin had a markedly lower affinity for the low-affinity site (pKd,lo = 5.29 and 4.58, respectively); chicken motilin had a lower affinity for both sites (pKd,hi = 8.36, pKd,lo = 3.97). Erythromycin A and its derivative N-trimethyl erythromycin A cnol ether also bound to cerebellar motilin receptors (pKd,hi = 7.29 and 8.91, respectively). Structure-activity studies with motilin fragments and the potency ranking of agonists suggest that a novel subtype receptor of motilin may exist in the brain. Guanosine 5'-O-(3-thiotriphosphate) (0.1 mM) reduced the number and the affinity for the high-affinity binding sites, which is evidence for G protein-coupled receptors. Our findings open new perspectives for the study of the physiological role of motilin.

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Investigating the conformational response of the Sortilin receptor upon binding endogenous peptide- and protein ligands by HDX-MS

10.1101/415620 ◽

2018 ◽

Author(s):

Esben Trabjerg ◽

Nadia Abu-Asad ◽

Ziqian Wan ◽

Fredrik Kartberg ◽

Søren Christensen ◽

...

Keyword(s):

Binding Sites ◽

Proximal Region ◽

Binding Region ◽

Exchange Mass Spectrometry ◽

Protein Ligands ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms ◽

Membrane Proximal Region

AbstractSortilin is a multifunctional transmembrane neuronal receptor involved in sorting of neurotrophic factors and apoptosis signalling. So far, structural characterization of Sortilin and its endogenous ligands has been limited to crystallographic studies of Sortilin in complex with the neuropeptide Neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of Sortilin to binding biological ligands including the peptides Neurotensin and the Sortilin propeptide and the proteins Progranulin and pro-Nerve growth factor-β. The results show that the ligands employ two binding sites inside the cavity of the β-propeller of Sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of Sortilin. Our findings improves our molecular understanding of Sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.

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A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010241118 ◽

2021 ◽

Vol 118 (8) ◽

pp. e2010241118

Author(s):

Tiantian Bu ◽

Sijia Lu ◽

Kai Wang ◽

Lidong Dong ◽

Shilin Li ◽

...

Keyword(s):

Binding Sites ◽

Crop Production ◽

Critical Role ◽

Photoperiod Sensitivity ◽

Low Latitude ◽

Flowering Phase ◽

Short Day ◽

Key Factor

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the “Maryland Mammoth” tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553–606 (1920)]. We further demonstrate that the J–LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC–E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

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