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Nucleotide Binding (nucleotide + binding)

Distribution by Scientific Domains
Chemistry66%
Medical Sciences16%
Life Sciences16%

Terms modified by Nucleotide Binding

  • nucleotide binding protein
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  • nucleotide binding site
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    Selected Abstracts

    Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides

    ACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010
    Matthias Haffke
    The human ATP-binding cassette (ABC) transporter ABCB6 is involved in haem-precursor transport across the mitochondrial membrane. The crystal structure of its nucleotide-binding domain (NBD) has been determined in the apo form and in complexes with ADP, with ADP and Mg2+ and with ATP at high resolution. The overall structure is L-shaped and consists of two lobes, consistent with other reported NBD structures. Nucleotide binding is mediated by the highly conserved Tyr599 and the Walker A motif, and induces notable structural changes. Structural comparison with other structurally characterized NBDs and full-length ABC transporters gives the first insight into the possible catalytic mechanism of ABCB6 and the role of the N-terminal helix ,1 in full-length ABCB6. [source]

    Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains

    FEBS JOURNAL, Issue 2 2007
    Anne R. Karow
    RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source]

    Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB)

    FEBS JOURNAL, Issue 15 2002
    2-APB reduces both Ca2+ binding, by interfering with the pathway leading to the Ca2+ -binding sites, phosphoryl transfer from ATP
    2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB onthe SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 µm, respectively, measured at pH 7.2). The Ca2+ -ATPase is also more potently inhibited at lower pH (IC50 = 70 µm for SERCA1A at pH 6). 2-APB decreases the affinity for Ca2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca2+ -ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca2+ to their binding sites. [source]

    Ribonucleotide activation by enzyme ribonucleotide reductase: Understanding the role of the enzyme

    JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 16 2004
    Nuno M. F. S. A. Cerqueira
    Abstract This article focuses on the first step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This corresponds to the activation of the substrate. In this work a large model of the active site region involving 130 atoms was used instead of the minimal gas phase models used in previous works. The ONIOM method was employed to deal with such a large system. The results gave additional information, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme in this step. Enzyme,substrate interaction energies, specific transition state stabilization, and substrate steric strain energies were obtained. It was concluded that the transition state is stabilized in 4.0 kcal/mol by specific enzyme,substrate interactions. However, this stabilization is cancelled by the cost in conformational energy for the enzyme to adopt the transition state geometry; the overall result is that the enzyme machinery does not lead to a rate enhancement in this step. It was also found that the substrate binds to the active site with almost no steric strain, emphasizing the complementarity and specificity of the RNR active site for nucleotide binding. The main role of the enzyme at the very beginning of the catalytic cycle was concluded to be to impose stereospecifity upon substrate activation and to protect the enzyme radical from the solvent, rather than to be an reaction rate enhancement. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 2031,2037, 2004 [source]

    A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

    ALLERGY, Issue 6 2009
    S. Salemi
    Background:, The Fip1-like-1,platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. Methods:, In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. Results:, Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. Conclusions:, We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias. [source]

    Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

    MOLECULAR MICROBIOLOGY, Issue 4 2001
    B. Lin
    The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector-binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant-GDP and mant-GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg-like protein results in the lack of function in vivo. [source]

    Energy-dependent degradation: Linkage between ClpX-catalyzed nucleotide hydrolysis and protein-substrate processing

    PROTEIN SCIENCE, Issue 5 2003
    Randall E. Burton
    Abstract ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATP,S by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATP,S to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. KM and kcat for hydrolysis of ATP and ATP,S were linearly correlated over a 200-fold range, suggesting that protein partners largely affect kcat rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of ,15 ,M on KD for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATP,S (,2 ,M) and ADP (,3 ,M) under the reaction conditions used for steady-state kinetics. In the absence of Mg2+, where hydrolysis does not occur, the inhibition constant for ATP (,55 ,M) was weaker but very similar to the value for ATP,S (,45 ,M). Compared with ATP, ATP,S supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis. [source]

    Mapping nucleotide binding site of calcium ATPase with IR spectroscopy: Effects of ATP ,-phosphate binding

    BIOPOLYMERS, Issue 4-5 2002
    Man Liu
    Abstract The changes in the IR spectra of the sarcoplasmic reticulum Ca2+ -ATPase upon nucleotide binding are recorded in H2O at 1°C in different buffers [imidazole, methylimidazole, 3-(N -morpholino)propanesulfonic acid, and phosphate] at different pH values (pH 6.5,7.8). The difference spectra of nucleotide binding are sensitive to the composition of the solvent. With methylimidazole at pH 7.5 providing the largest binding-induced signals, the effects of ,-phosphate binding are investigated using ATP, ADP, and ,,,-iminoadenosine 5,-triphosphate. The ,-phosphate contributes ,20% to the conformational change seen by IR spectroscopy and affects the ,-sheet structures. The IR experiments also reveal the known affinity difference between ADP and ATP. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 67: 267,270, 2002 [source]

    Regulation and Role of the Presynaptic and Myocardial Na+/H+ Exchanger NHE1: Effects on the Sympathetic Nervous System in Heart Failure

    CARDIOVASCULAR THERAPEUTICS, Issue 2 2007
    Kirsten Leineweber
    ABSTRACT In acute myocardial ischemia and in chronic heart failure, sympathetic activation with excessive norepinephrine (NE) release from and reduced NE reuptake into sympathetic nerve endings is a prominent cause of arrhythmias and cardiac dysfunction. The Na+/H+ exchanger NHE1 is the predominant isoform in the heart. It contributes to cellular acid,base balance, and electrolyte, and volume homeostasis, and is activated in response to intracellular acidosis and/or activation of guanine nucleotide binding (G) protein-coupled receptors. NHE1 mediates its signaling via protein kinases A (PKA) or C (PKC). In cardiomyocytes, NHE1 is restricted to specialized membrane domains, where it regulates the activity of pH-sensitive proteins and modulates the driving force of the Na+/Ca2+ exchanger. During acute ischemia/reperfusion and in heart failure the activity/amount of NHE1 is increased, leading to intracellular Ca2+ overload and promoting structural (apoptosis, hypertrophy) and functional (arrhythmias, hypercontraction) myocardial damage. In sympathetic nerve endings, increased NHE1 activity results in the accumulation of axoplasmic Na+ that diminishes the inward and/or favors the outward transport of NE via the neuronal norepinephrine transporter (NET). The increased NE levels within the nerve,muscle junction facilitate the sustained stimulation of myocardial ,- and ,-adrenoceptors (ARs), which in turn aggravate the increases in myocardial NHE1 activity and the associated deleterious effects. Furthermore, the responsiveness of the ,-AR declines overtime, which results in further release of NE, initiating a vicious cycle. Accordingly, NHE1 is a potential candidate for targeted intervention to suppress this feedback loop. [source]

    Mimicking small G-proteins: an emerging theme from the bacterial virulence arsenal

    CELLULAR MICROBIOLOGY, Issue 3 2008
    Neal M. Alto
    Summary The identification of the Ras superfamily of small molecular weight GTPases (G-proteins) has opened up new fields in cancer biology, immunity and infectious disease research. Because of their ubiquitous role in cellular homeostasis, small G-proteins are common targets for several pathogens, including bacteria. It is well known that pathogenic bacteria have evolved virulence factors that chemically modify GTPases or directly mimic the activities of key regulatory proteins. However, recent studies now suggest that bacterial ,effector' proteins can also mimic the activities of Ras small G-proteins despite their lack of guanine nucleotide binding or GTPase enzymatic activity. The study of these unique pathogenic strategies continues to reveal novel mechanistic insights into host cellular communication networks and the role of small G-protein signalling during human infectious disease. [source]

    A MAS NMR Study of the Bacterial ABC Transporter ArtMP

    CHEMBIOCHEM, Issue 4 2010
    Vivien Lange Dr.
    Abstract ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively 13C,15N-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment. [source]

    Hydrogen-Bond-Guided Self-Assembly of Nucleotides on a Receptor-Array Surface

    CHEMISTRY - A EUROPEAN JOURNAL, Issue 34 2010
    Dr. Dmitry
    Abstract The hydrogen-bond-guided self-assembly of 5,-ribonucleotides bearing adenine(A), cytosine (C), uracil (U), or guanine (G) bases from aqueous solution on a lipid-like surface decorated with synthetic bis(ZnII,cyclen) (cyclen=1,4,7,10-tetraazacyclodododecane) metal,complex receptor sites is described. The process was studied by using surface plasmon resonance spectroscopy. The data show that the mechanism of nucleotide binding to the 2D template is influenced by the chemistry of the bases and the pH,value of the solution. In a neutral solution of pH,7.5, the process is cooperative and selective with respect to Watson,Crick pairs (A,U and C,G), which form stable double planes in accordance with the Chargaff rule. In a more acidic solution at pH,6.0, the interactions between complementary partners become non-cooperative and the surface also stabilizes mismatched and wobble pairs due to the pH-induced changes in the receptor coordination state. The results suggest that hydrogen bonding plays a key role in the self-assembly of complementary nucleotides at the lipid-like interface, and the cooperative character of the process stems from the ideal matching of the orientation and chemistry of all the interacting components with respect to each other in neutral solution. [source]