Journal articles on the topic 'Nucleotides binding'
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1
McIlroy, Patrick J. "Effects of cations on binding of human choriogonadotropin." Biochemistry and Cell Biology 66, no. 12 (December 1, 1988): 1258–64. http://dx.doi.org/10.1139/o88-145.
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The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.
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Hause, Lara L., and Kevin S. McIver. "Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences." Journal of Bacteriology 194, no. 18 (July 6, 2012): 4904–19. http://dx.doi.org/10.1128/jb.00809-12.
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ABSTRACTThe Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.
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Chander, Preethi, Kari M. Halbig, Jamie K. Miller, Christopher J. Fields, Heather K. S. Bonner, Gail K. Grabner, Robert L. Switzer, and Janet L. Smith. "Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus, Suggests Dual Regulation by Pyrimidine and Purine Nucleotides." Journal of Bacteriology 187, no. 5 (March 1, 2005): 1773–82. http://dx.doi.org/10.1128/jb.187.5.1773-1782.2005.
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ABSTRACT PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60°C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg2+, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg2+ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.
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Schulz, Georg E. "Binding of nucleotides by proteins." Current Opinion in Structural Biology 2, no. 1 (February 1992): 61–67. http://dx.doi.org/10.1016/0959-440x(92)90178-a.
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Schulz, Georg E. "Binding of nucleotides by proteins." Current Biology 2, no. 2 (February 1992): 81. http://dx.doi.org/10.1016/0960-9822(92)90208-r.
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Csanády, László, and Vera Adam-Vizi. "Antagonistic Regulation of Native Ca2+- and ATP-sensitive Cation Channels in Brain Capillaries by Nucleotides and Decavanadate." Journal of General Physiology 123, no. 6 (June 1, 2004): 743–57. http://dx.doi.org/10.1085/jgp.200309008.
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Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 μM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3′-5′ cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned channels.
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Sullivan, S. M., R. Mishra, R. R. Neubig, and J. R. Maddock. "Analysis of Guanine Nucleotide Binding and Exchange Kinetics of the Escherichia coli GTPase Era." Journal of Bacteriology 182, no. 12 (June 15, 2000): 3460–66. http://dx.doi.org/10.1128/jb.182.12.3460-3466.2000.
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ABSTRACT Era is an essential Escherichia coli guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (N-methyl-3′-O-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (KD ) for mGDP and mGTP (0.61 ± 0.12 μM and 3.6 ± 0.80 μM, respectively) are similar to those of the unmodified nucleotides. The single turnover rates for mGTP hydrolysis by Era were 3.1 ± 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl2 and 5.6 ± 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl2. Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg2+. We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.
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Lew, Chih M., and Jay D. Gralla. "Mechanism of Stimulation of Ribosomal Promoters by Binding of the +1 and +2 Nucleotides." Journal of Biological Chemistry 279, no. 19 (March 9, 2004): 19481–85. http://dx.doi.org/10.1074/jbc.m401285200.
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The rate of transcription ofEscherichia coliribosomal RNA promoters is central to adjusting the cellular growth rate to nutritional conditions. The +1 initiating nucleotide and ppGpp are regulatory effectors of these promoters. The data herein show thatin vitrotranscription is also regulated by the +2 nucleotide. Both the +1 and +2 nucleotides act by driving polymerase into an altered conformation rather than by increasing the lifetime of transcription complexes. The unique design of the ribosomal promoters may stabilize a distorted state of polymerase that is relieved by the binding of the two nucleotides required for transcription initiation.
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Gromadski, Kirill B., Tobias Schümmer, Anne Strømgaard, Charlotte R. Knudsen, Terri Goss Kinzy, and Marina V. Rodnina. "Kinetics of the Interactions between Yeast Elongation Factors 1A and 1Bα, Guanine Nucleotides, and Aminoacyl-tRNA." Journal of Biological Chemistry 282, no. 49 (October 9, 2007): 35629–37. http://dx.doi.org/10.1074/jbc.m707245200.
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The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1Bα (eEF1Bα), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 μm, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s–1) and is accelerated by eEF1Bα by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1Bα binding to eEF1A (107–108m–1 s–1) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s–1, which is compatible with the rate of protein synthesis in the cell. eEF1A·GTP binds Phe-tRNAPhe with a Kd of 3 nm, whereas eEF1A·GDP shows no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu.
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Demeuse, Philippe, Reinhold Penner, and Andrea Fleig. "TRPM7 Channel Is Regulated by Magnesium Nucleotides via its Kinase Domain." Journal of General Physiology 127, no. 4 (March 13, 2006): 421–34. http://dx.doi.org/10.1085/jgp.200509410.
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TRPM7 is a Ca2+- and Mg2+-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg2+, the functional roles of intracellular levels of Mg·ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg·ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg2+. Mg·ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg2+ and Ca2+ overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP ≥ GTP ≥ UTP > ITP ≈ free Mg2+ alone. These nucleotides also enhanced TRPM7 inhibition by free Mg2+, suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg2+-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg·NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg2+ binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg2+ binding site extrinsic to that domain.
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Mortensen, E. R., J. Drachman, and G. Guidotti. "Guanosine nucleotides regulate hormone binding of insulin receptors." Biochemical Journal 281, no. 3 (February 1, 1992): 735–43. http://dx.doi.org/10.1042/bj2810735.
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Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.
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Liu, Qi, Reed F. Johnson, and Julian L. Leibowitz. "Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA." Journal of Virology 75, no. 24 (December 15, 2001): 12105–13. http://dx.doi.org/10.1128/jvi.75.24.12105-12113.2001.
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ABSTRACT Previously, we characterized two host protein binding elements located within the 3′-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3′-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).
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Eraso, Jesus M., and Samuel Kaplan. "Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1." Journal of Bacteriology 191, no. 13 (May 1, 2009): 4353–64. http://dx.doi.org/10.1128/jb.00244-09.
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ABSTRACT The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.
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Sun, Jie, and Ke Chen. "NSiteMatch: Prediction of Binding Sites of Nucleotides by Identifying the Structure Similarity of Local Surface Patches." Computational and Mathematical Methods in Medicine 2017 (2017): 1–16. http://dx.doi.org/10.1155/2017/5471607.
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Nucleotides play a central role in life-form metabolism, by interacting with proteins and mediating the function of proteins. It is estimated that nucleotides constitute about 15% of the biologically relevant ligands included in PDB. Prediction of binding sites of nucleotides is useful in understanding the function of proteins and can facilitate the in silico design of drugs. In this study, we propose a nucleotide-binding site predictor, namely, NSiteMatch. The NSiteMatch algorithm integrates three different strategies: geometrical analysis, energy calculation, and template comparison. Unlike a traditional template-based predictor, which identifies global similarity between target structure and template, NSiteMatch concerns the local similarity between a surface patch of the target protein and the binding sites of template. To this end, NSiteMatch identifies more templates than traditional template-based predictors. The NSiteMatch predictor is compared with three representative methods, Findsite, Q-SiteFinder, and MetaPocket. An extensive evaluation demonstrates that NSiteMatch achieves higher success rates than Findsite, Q-SiteFinder, and MetaPocket, in prediction of binding sites of ATP, ADP, and AMP.
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Weinreich, Frank, John R. Riordan, and Georg Nagel. "Dual Effects of Adp and Adenylylimidodiphosphate on Cftr Channel Kinetics Show Binding to Two Different Nucleotide Binding Sites." Journal of General Physiology 114, no. 1 (July 1, 1999): 55–70. http://dx.doi.org/10.1085/jgp.114.1.55.
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The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.
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WANG, Guichun, Roxana PINCHEIRA, Mei ZHANG, and Jian-Ting ZHANG. "Conformational changes of P-glycoprotein by nucleotide binding." Biochemical Journal 328, no. 3 (December 15, 1997): 897–904. http://dx.doi.org/10.1042/bj3280897.
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P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5ʹ-[β,γ-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport.
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Tuvshinjargal, Narankhuu, Wook Lee, Byungkyu Park, and Kyungsook Han. "Predicting protein-binding RNA nucleotides with consideration of binding partners." Computer Methods and Programs in Biomedicine 120, no. 1 (June 2015): 3–15. http://dx.doi.org/10.1016/j.cmpb.2015.03.010.
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Dawicki, D. D., J. McGowan-Jordan, S. Bullard, S. Pond, and S. Rounds. "Extracellular nucleotides stimulate leukocyte adherence to cultured pulmonary artery endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 4 (April 1, 1995): L666—L673. http://dx.doi.org/10.1152/ajplung.1995.268.4.l666.
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Adenosine, ATP, and various nucleotides were examined for their effects on the adherence of leukocytes to bovine pulmonary artery endothelial cells. Extracellular ATP enhanced adherence of HL-60 cells and human neutrophils to endothelial cells in a dose-dependent fashion. Maximal adherence occurred after 15 min coincubation of ATP and HL-60 cells or neutrophils with endothelial cells. ATP stimulation was mediated by direct effects on both HL-60 cells and endothelial cells. The potency profile of various nucleotides was ATP = 2-MeSATP > beta,gamma-CH2ATP, indicative of a P2y receptor. Interestingly, UTP was as potent as ATP in stimulating HL-60 cell adherence, suggesting the presence of a pyrimidine nucleotide receptor. Photoaffinity labeling of endothelial cells with 8-Az-[alpha-32P]ATP showed the presence of two ATP binding proteins of 48 and 87 kDa. ATP and 2-MeSATP inhibited binding by both proteins. Labeling of the 87-kDa protein was inhibited by beta,gamma-CH2ATP, whereas UTP blocked binding by the 48-kDa protein. Thus photoaffinity labeling experiments support the proposal that endothelial cells possess two ATP receptors, one of which is a P2u nucleotide receptor. These findings show that extracellular nucleotides enhance leukocyte adherence to endothelial cells. Nucleotide release into the extracellular space may be one mechanism of exacerbating vascular cell injury relevant to conditions such as adult respiratory distress syndrome and septic shock.
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Katwa, L. C., C. D. Parker, J. K. Dybing, and A. A. White. "Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation." Biochemical Journal 283, no. 3 (May 1, 1992): 727–35. http://dx.doi.org/10.1042/bj2830727.
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Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes. ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM. The strongest inhibitors were adenosine 5′-[beta gamma-imido]triphosphate (App[NH]p) (36%) and adenosine 5′-[beta-thio]diphosphate (ADP[S]) (41%). Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS). Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP[S] (41%). While App[NH]p and MgGDP[S] appeared to be acting at different sites, they also interfered with each other. These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase. Inhibition by low concentrations of MgApp[NH]p and MgATP was weaker above 0.1 mM, while MgGDP[S] and magnesium guanosine 5′-[gamma-thio]triphosphate (MgGTP[S]) inhibited in a single phase. Inhibition by MgApp[NH]p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP[S] and MgGTP[S]. Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP. This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP. These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation.
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Huang, Ji, Vinh H. Nguyen, Karleigh A. Hamblin, Robin Maytum, Mark van der Giezen, and Marie E. Fraser. "ATP-specificity of succinyl-CoA synthetase fromBlastocystis hominis." Acta Crystallographica Section D Structural Biology 75, no. 7 (June 26, 2019): 647–59. http://dx.doi.org/10.1107/s2059798319007976.
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Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46βE, K114βD, V113βL and L227βF, and one with two mutations, K46βE/K114βD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20β, Gly111β and Val113β. The O atom of the side chain of Gln20β interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111β. It is the different conformations of the backbone at Gln20β, of the side chain of Gln20β and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227βF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46βE/K114βD double mutant ofBlastocystis hominisSCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.
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Ohmori, Senri, Marina Wani, Saki Kitabatake, Yuka Nakatsugawa, Tadashi Ando, Takuya Umehara, and Koji Tamura. "RNA Aptamers for a tRNA-Binding Protein from Aeropyrum pernix with Homologous Counterparts Distributed Throughout Evolution." Life 10, no. 2 (February 1, 2020): 11. http://dx.doi.org/10.3390/life10020011.
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In the present in vitro selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon Aeropyrum pernix. Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for A. pernix Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3′-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3′ terminus of tRNA in the interaction with Trbp-like structures.
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Hosoi, K., M. Fujishita, K. Sugita, K. Kurihara, T. Atsumi, T. Murai, and T. Ueha. "P2 purinergic receptors and cellular calcium metabolism in A 431 human epidermoid carcinoma cells." American Journal of Physiology-Cell Physiology 262, no. 3 (March 1, 1992): C635—C643. http://dx.doi.org/10.1152/ajpcell.1992.262.3.c635.
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Stimulation of P2 purinergic receptors on A 431 human epidermoid cells with ATP rapidly mobilized intracellular calcium and increased cytosolic free Ca2+ ([Ca2+]i). Incorporation of 45Ca2+ was also stimulated by ATP at a rate less than that of [Ca2+]i elevation. Among a number of nucleosides, nucleotides, and their analogues examined, ATP, GTP, UTP, ADP, UDP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and 5'-adenylylimidodiphosphate (AMP-PNP) increased both [Ca2+]i and 45Ca2+ influx, whereas others did not; these latter two analogues (ATP gamma S and AMP-PNP) blocked the ATP-stimulated 45Ca2+ influx only very slightly, suggesting that they are not prominent antagonists but rather agonists. A high correlation between [Ca2+]i increase and 45Ca2+ influx, in terms of nucleotide specificity, suggests the involvement of [Ca2+]i in influx of 45Ca2+. It appeared that [Ca2+]i elevated by several nucleotides or nucleotide analogues opened a calcium gate, thus allowing the influx of 45Ca2+. P2 purinergic receptors on these cells had such a characteristic that they were rapidly desensitized. These nucleotides or analogues also affected epidermal growth factor (EGF) receptors by inhibiting the EGF binding. The differences of ligand or substrate specificities between P2 purinergic receptors and ecto-nucleotidases indicates that the two components are different molecules involved in different systems.
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Beslin, A., M. P. Vié, J. P. Blondeau, and J. Francon. "Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells." Biochemical Journal 305, no. 3 (February 1, 1995): 729–37. http://dx.doi.org/10.1042/bj3050729.
Full textAbstract:
High-affinity 3,3′,5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3′,5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3′5′-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).
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Cameron, Angus J. M. "Occupational hazards: allosteric regulation of protein kinases through the nucleotide-binding pocket." Biochemical Society Transactions 39, no. 2 (March 22, 2011): 472–76. http://dx.doi.org/10.1042/bst0390472.
Full textAbstract:
Targeting the protein kinase ATP-binding pocket provides a significant opportunity for the treatment of disease. Recent studies have revealed a central activity-independent role for nucleotide pocket occupation in the allosteric behaviour of diverse kinases. Regulation of nucleotide pocket conformation with either nucleotides or ATP competitive inhibitors has revealed an added dimension to the targeting of kinases. In the present paper, using PKC (protein kinase C) as a paradigm, the liabilities and opportunities associated with the occupation of the nucleotide pocket are explored.
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Dolgounitcheva, O., V. G. Zakrzewski, and J. V. Ortiz. "Electron binding energies of nucleobases and nucleotides." International Journal of Quantum Chemistry 90, no. 4-5 (2002): 1547–54. http://dx.doi.org/10.1002/qua.10380.
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Matthiesen, Karina, and Jacob Nielsen. "Binding of cyclic nucleotides to phosphodiesterase 10A and 11A GAF domains does not stimulate catalytic activity." Biochemical Journal 423, no. 3 (October 12, 2009): 401–9. http://dx.doi.org/10.1042/bj20090982.
Full textAbstract:
To date eleven human PDE (3′,5′-cyclic nucleotide phosphodiesterase) families have been identified. Of these, five families contain non-catalytic tandem GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaenaadenylate cyclases and Escherichia coliFhlA) domains, GAFa and GAFb, in the N-terminal part of the enzyme. For PDE2A, PDE5A and PDE6 the GAF domains have been shown to bind cGMP with high affinity. For PDE2A and PDE5A this ligand binding has been shown to stimulate the catalytic activity of the enzyme. PDE10A and PDE11A are the two most recently described PDEs and it has been suggested that their GAF domains bind to cAMP and cGMP respectively. We have developed a scintillation proximity-based assay to directly measure cyclic nucleotide binding to the PDE2A, PDE10A and PDE11A GAF domains, and in the present study we demonstrate binding of cyclic nucleotides to the PDE10A and PDE11A GAF domains. We show that these non-catalytic sites bind cAMP and cGMP respectively with much higher affinity than has previously been suggested using indirect assessment of the interaction. The GAFb domain of PDE10A binds cAMP with a Kd of 48 nM and the GAFa domain of PDE11A binds cGMP with a Kd of 110 nM. The effect of cyclic nucleotides binding to the GAF domains on the enzyme activity was investigated through the use of modified cyclic nucleotides. In contrast with other GAF domain-containing PDEs, and with what has previously been predicted, ligand binding to the GAF domains of PDE10A and PDE11A does not stimulate catalytic activity.
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Mathis, S. A., and L. M. F. Leeb-Lundberg. "Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides." Biochemical Journal 276, no. 1 (May 15, 1991): 141–47. http://dx.doi.org/10.1042/bj2760141.
Full textAbstract:
We have previously reported that [3H]bradykinin [(3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (Bmax) of 1119 +/- 160 fmol/mg of protein, with an equilibrium dissociation constant (KD) of 314 +/- 70 pM and with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5′[beta gamma-imido]triphosphate (Gpp[NH]p, [3H]BK bound to the soluble receptors with a KD of 929 +/- 129 pM and a Bmax. of 706 +/- 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and Bmax., which was half-maximal at 0.5 microM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G-)protein.
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Query, C. C., R. C. Bentley, and J. D. Keene. "A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component." Molecular and Cellular Biology 9, no. 11 (November 1989): 4872–81. http://dx.doi.org/10.1128/mcb.9.11.4872.
Full textAbstract:
We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.
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Query, C. C., R. C. Bentley, and J. D. Keene. "A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component." Molecular and Cellular Biology 9, no. 11 (November 1989): 4872–81. http://dx.doi.org/10.1128/mcb.9.11.4872-4881.1989.
Full textAbstract:
We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.
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Frey, Stephan, Adriane Leskovar, Jochen Reinstein, and Johannes Buchner. "The ATPase Cycle of the Endoplasmic Chaperone Grp94." Journal of Biological Chemistry 282, no. 49 (October 9, 2007): 35612–20. http://dx.doi.org/10.1074/jbc.m704647200.
Full textAbstract:
Grp94, the Hsp90 paralog of the endoplasmic reticulum, plays a crucial role in protein secretion. Like cytoplasmic Hsp90, Grp94 is regulated by nucleotide binding to its N-terminal domain. However, the question of whether Grp94 hydrolyzes ATP was controversial. This sets Grp94 apart from other members of the Hsp90 family where a slow but specific turnover of ATP has been unambiguously established. In this study we aimed at analyzing the nucleotide binding properties and the potential ATPase activity of Grp94. We show here that Grp94 has an ATPase activity comparable with that of yeast Hsp90 with a kcat of 0.36 min–1 at 25 °C. Kinetic and equilibrium constants of the partial reactions of the ATPase cycle were determined using transient kinetic methods. Nucleotide binding appears to be tighter compared with other Hsp90s investigated, with dissociation constants (KD) of ∼4 μm for ADP, ATP, and AMP-PCP. Interestingly, all nucleotides and inhibitors (radicicol, 5′-N-ethylcarboxamidoadenosine) studied here bind with similar rate constants for association (0.2–0.3 × 106m–1 s–1). Furthermore, there is a marked difference from cytosolic Hsp90s in that after binding, the ATP molecule does not seem to become trapped by conformational changes in Grp94. Grp94 stays predominantly in the open state concerning the nucleotide-binding pocket as evidenced by kinetic analyses. Thus, Grp94 shows mechanistically important differences in the interaction with adenosine nucleotides, but the basic hydrolysis reaction seems to be conserved between cytosolic and endoplasmic members of the Hsp90 family.
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Bag, Jnanankur. "Feedback Inhibition of Poly(A)-binding Protein mRNA Translation." Journal of Biological Chemistry 276, no. 50 (October 4, 2001): 47352–60. http://dx.doi.org/10.1074/jbc.m107676200.
Full textAbstract:
An adenine-richciselement at the 5′-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter β-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5′-UTR of this mRNA. For this translational control the distance of the ARS from the 5′ cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5′-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5′-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5′ cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5′ cap to a distance of ∼267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.
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Volkán-Kacsó, Sándor, and Rudolph A. Marcus. "Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): 12029–34. http://dx.doi.org/10.1073/pnas.1611601113.
Full textAbstract:
A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F1-ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.
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BISWAS, Subhasis B., Stephen FLOWERS, and Esther E. BISWAS-FISS. "Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli." Biochemical Journal 379, no. 3 (May 1, 2004): 553–62. http://dx.doi.org/10.1042/bj20031255.
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In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC–DNA and DnaC–DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6•DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.
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Hiriyanna, K. T., J. Varkey, M. Beer, and R. M. Benbow. "Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo." Journal of Cell Biology 107, no. 1 (July 1, 1988): 33–44. http://dx.doi.org/10.1083/jcb.107.1.33.
Full textAbstract:
Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy-nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.
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KULAKOVSKIY, IVAN, VICTOR LEVITSKY, DMITRY OSHCHEPKOV, LEONID BRYZGALOV, ILYA VORONTSOV, and VSEVOLOD MAKEEV. "FROM BINDING MOTIFS IN CHIP-SEQ DATA TO IMPROVED MODELS OF TRANSCRIPTION FACTOR BINDING SITES." Journal of Bioinformatics and Computational Biology 11, no. 01 (February 2013): 1340004. http://dx.doi.org/10.1142/s0219720013400040.
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Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) became a method of choice to locate DNA segments bound by different regulatory proteins. ChIP-Seq produces extremely valuable information to study transcriptional regulation. The wet-lab workflow is often supported by downstream computational analysis including construction of models of nucleotide sequences of transcription factor binding sites in DNA, which can be used to detect binding sites in ChIP-Seq data at a single base pair resolution. The most popular TFBS model is represented by positional weight matrix (PWM) with statistically independent positional weights of nucleotides in different columns; such PWMs are constructed from a gapless multiple local alignment of sequences containing experimentally identified TFBSs. Modern high-throughput techniques, including ChIP-Seq, provide enough data for careful training of advanced models containing more parameters than PWM. Yet, many suggested multiparametric models often provide only incremental improvement of TFBS recognition quality comparing to traditional PWMs trained on ChIP-Seq data. We present a novel computational tool, diChIPMunk, that constructs TFBS models as optimal dinucleotide PWMs, thus accounting for correlations between nucleotides neighboring in input sequences. diChIPMunk utilizes many advantages of ChIPMunk, its ancestor algorithm, accounting for ChIP-Seq base coverage profiles ("peak shape") and using the effective subsampling-based core procedure which allows processing of large datasets. We demonstrate that diPWMs constructed by diChIPMunk outperform traditional PWMs constructed by ChIPMunk from the same ChIP-Seq data. Software website: http://autosome.ru/dichipmunk/
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Brown, Jessica A., Likui Zhang, Shanen M. Sherrer, John-Stephen Taylor, Peter M. J. Burgers, and Zucai Suo. "Pre-Steady-State Kinetic Analysis of Truncated and Full-LengthSaccharomyces cerevisiaeDNA Polymerase Eta." Journal of Nucleic Acids 2010 (2010): 1–11. http://dx.doi.org/10.4061/2010/871939.
Full textAbstract:
Understanding polymerase fidelity is an important objective towards ascertaining the overall stability of an organism's genome.Saccharomyces cerevisiaeDNA polymeraseη(yPolη), a Y-family DNA polymerase, is known to efficiently bypass DNA lesions (e.g., pyrimidine dimers) in vivo. Using pre-steady-state kinetic methods, we examined both full-length and a truncated version of yPolηwhich contains only the polymerase domain. In the absence of yPolη's C-terminal residues 514–632, the DNA binding affinity was weakened by 2-fold and the base substitution fidelity dropped by 3-fold. Thus, the C-terminus of yPolηmay interact with DNA and slightly alter the conformation of the polymerase domain during catalysis. In general, yPolηdiscriminated between a correct and incorrect nucleotide more during the incorporation step (50-fold on average) than the ground-state binding step (18-fold on average). Blunt-end additions of dATP or pyrene nucleotide5′-triphosphate revealed the importance of base stacking during the binding of incorrect incoming nucleotides.
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QIN, PENG-HUA, WEN-CAI LU, PAN-JUAN GUO, WEI QIN, LI-ZHEN ZHAO, and WEI SONG. "INTERACTIONS OF THE NUCLEOTIDES WITH THE METAL IONS Mg2+, Ca2+, Mn2+, Na+, AND K+." Journal of Theoretical and Computational Chemistry 11, no. 06 (December 2012): 1183–99. http://dx.doi.org/10.1142/s0219633612500794.
Full textAbstract:
The interactions of the four typical nucleotides with the metal ions Mg 2+, Ca 2+, Mn 2+, Na +, and K + were studied by using the B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p) calculations in the PCM model. A lot of initial binding sites of the metal ions were designed and optimized to determine the most stable structures of the metal ion nucleotide compounds. It has been shown that the metal ions tend to attach at the center of the negatively charged atoms of the nucleotides. Furthermore, the vertical excitation energies of the metal ion nucleotide compounds were calculated at the same level with the TDDFT method, and also NBO charges were analyzed to understand the bonding characteristics between the metal ions and the nucleotides. That was also compared with the conclusion in the gas phase.
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MISSIAEN, Ludwig, Jan B. PARYS, Humbert DE SMEDT, Ilse SIENAERT, Henk SIPMA, Sara VANLINGEN, Karlien MAES, and Rik CASTEELS. "Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release." Biochemical Journal 325, no. 3 (August 1, 1997): 661–66. http://dx.doi.org/10.1042/bj3250661.
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The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1,4,5)P3 receptor. It was observed that 3′-phosphoadenosine 5′-phosphosulphate, CoA, di(adenosine-5′)tetraphosphate (Ap4A) and di(adenosine-5′)pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4,5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.
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Sakamoto, C., T. Matozaki, M. Nagao, and S. Baba. "Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 253, no. 3 (September 1, 1987): G308—G314. http://dx.doi.org/10.1152/ajpgi.1987.253.3.g308.
Full textAbstract:
Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (Ni) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125I-[Tyr1]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p] greater than GTP greater than GDP greater than GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg2+. When pancreatic acini were treated with 1 microgram/ml pertussis toxin for 4 h, subsequent 125I-[Tyr1]somatostatin binding to acinar membranes was reduced. Gpp(NH)p further decreased somatostatin binding to islet-activating protein (IAP)-treated acinar membranes. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. Furthermore, exposure of acini to IAP caused ADP ribosylation of a membrane protein with Mr = 41,000 in parallel to the inhibition of cAMP accumulation in acini. The present results suggest, therefore, that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor.
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Proks, Peter, Heidi de Wet, and Frances M. Ashcroft. "Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: A mechanistic study." Journal of General Physiology 144, no. 5 (October 27, 2014): 469–86. http://dx.doi.org/10.1085/jgp.201411222.
Full textAbstract:
Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K+ (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.
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Stawicki, Scott Stevenson, and C. Cheng Kao. "Spatial Perturbations within an RNA Promoter Specifically Recognized by a Viral RNA-Dependent RNA Polymerase (RdRp) Reveal That RdRp Can Adjust Its Promoter Binding Sites." Journal of Virology 73, no. 1 (January 1, 1999): 198–204. http://dx.doi.org/10.1128/jvi.73.1.198-204.1999.
Full textAbstract:
ABSTRACT RNA synthesis during viral replication requires specific recognition of RNA promoters by the viral RNA-dependent RNA polymerase (RdRp). Four nucleotides (−17, −14, −13, and −11) within the brome mosaic virus (BMV) subgenomic core promoter are required for RNA synthesis by the BMV RdRp (R. W. Siegel et al., Proc. Natl. Acad. Sci. USA 94:11238–11243, 1997). The spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in RNAs containing nucleotide insertions and deletions within the BMV subgenomic core promoter. Spatial perturbations between nucleotides −17 and −11 resulted in decreased RNA synthesis in vitro. However, synthesis was still dependent on the key nucleotides identified in the wild-type core promoter and the initiation cytidylate. In contrast, changes between nucleotides −11 and +1 had a less severe effect on RNA synthesis but resulted in RNA products initiated at alternative locations in addition to the +1 cytidylate. The results suggest a degree of flexibility in the recognition of the subgenomic promoter by the BMV RdRp and are compared with functional regions in other DNA and RNA promoters.
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Lee, John H., and Randall K. Holmes. "Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength." Journal of Bacteriology 182, no. 2 (January 15, 2000): 432–38. http://dx.doi.org/10.1128/jb.182.2.432-438.2000.
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ABSTRACT The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe2+ as a corepressor. Holo-DtxR inhibits transcription from the iron-regulated promoters (IRPs) designated IRP1 through IRP5 as well as from the promoters for thetox and hmuO genes. DtxR binds to 19-bp operators with the consensus sequence 5′-TTAGGTTAGCCTAACCTAA-3′, a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5′-TTAGGTGAGACGCACCCAT-3′ [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5′ end with the putative −10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or −7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(−7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(−7) except for A(−7)C dramatically decreased IRP3 promoter strength. In contrast, the A(−7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5′-TTAGGTTAGCCAAACCTTT-3′) includes the −35 region of the IRP3 promoter. A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and −7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have significant effects on promoter activity for IRP3 and IRP1.
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Li, Yuan-Zong, Wen-Bao Chang, and Yun-Xiang Ci. "Structure effect of nucleotides in terbium(III)-nucleotide fluorescent reaction New evidence for the binding sites of terbium(III) on nucleotides." Chinese Journal of Chemistry 11, no. 6 (August 27, 2010): 524–31. http://dx.doi.org/10.1002/cjoc.19930110606.
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Burstein, E. S., and I. G. Macara. "Interactions of the ras-like protein p25rab 3A with Mg2+ and guanine nucleotides." Biochemical Journal 282, no. 2 (March 1, 1992): 387–92. http://dx.doi.org/10.1042/bj2820387.
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The rab3A gene product is a 25 kDa guanine-nucleotide-binding protein which is expressed at high levels in neural tissue and has about 30% sequence similarity to ras. Purified p25rab3A has been used as substrate to examine its kinetics of nucleotide binding and hydrolysis, and to study the effects of Mg2+ on these processes. p25rab3A binds GDP and GTP similarly well, with nanomolar affinity. Mg2+ increases the affinity between p25rab3A and guanine nucleotides by 3- and 7-fold for GTP and GDP respectively, primarily by drastically decreasing the nucleotide off-rates. The Mg2+ binding affinity to p25rab3A. [alpha 32P]GDP was determined to be about 4 microns using entrapment of [alpha-32P]GDP as a measure of Mg2+ binding. At a Mg2+ concentration of 11 mM. GTPase activity was rate-limited by the GDP off-rate. Surprisingly, at a Mg2+ concentration of 80 nM. GTPase activity was comparable with that in the presence of excess Mg2+. In this case, kcat. was rate-limiting. At Mg2+ concentrations below 10 nM there was no detectable GTPase activity, indicating that Mg2+ is required for the GTPase activity of p25rab3A.
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Xu, Chaoyi, Douglas K. Fischer, Sanela Rankovic, Wen Li, Robert A. Dick, Brent Runge, Roman Zadorozhnyi, et al. "Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis." PLOS Biology 18, no. 12 (December 17, 2020): e3001015. http://dx.doi.org/10.1371/journal.pbio.3001015.
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Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.
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Reiber, Duane C., and Robert C. Murphy. "Covalent Binding of LTA4 to Nucleosides and Nucleotides." Archives of Biochemistry and Biophysics 379, no. 1 (July 2000): 119–26. http://dx.doi.org/10.1006/abbi.2000.1851.
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Mejillano, Magdalena R., and Richard H. Himes. "Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site." Archives of Biochemistry and Biophysics 291, no. 2 (December 1991): 356–62. http://dx.doi.org/10.1016/0003-9861(91)90146-a.
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Haffke, Matthias, Anja Menzel, Yvonne Carius, Dieter Jahn, and Dirk W. Heinz. "Structures of the nucleotide-binding domain of the human ABCB6 transporter and its complexes with nucleotides." Acta Crystallographica Section D Biological Crystallography 66, no. 9 (August 13, 2010): 979–87. http://dx.doi.org/10.1107/s0907444910028593.
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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 α1 in full-length ABCB6.
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Sage, Jay M., Anthony J. Cura, Kenneth P. Lloyd, and Anthony Carruthers. "Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site." American Journal of Physiology-Cell Physiology 308, no. 10 (May 15, 2015): C827—C834. http://dx.doi.org/10.1152/ajpcell.00001.2015.
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Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3- O-methylglucose uptake in human erythrocytes [ Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3- O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites—the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.
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Coin, Frédéric, Philippe Frit, Benoit Viollet, Bernard Salles, and Jean-Marc Egly. "TATA Binding Protein Discriminates between Different Lesions on DNA, Resulting in a Transcription Decrease." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3907–14. http://dx.doi.org/10.1128/mcb.18.7.3907.
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ABSTRACT DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position −29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage.