scholarly journals Bfd, a New Class of [2Fe-2S] Protein That Functions in Bacterial Iron Homeostasis, Requires a Structural Anion Binding Site

Biochemistry ◽  
2018 ◽  
Vol 57 (38) ◽  
pp. 5533-5543 ◽  
Author(s):  
Harshani Wijerathne ◽  
Huili Yao ◽  
Yan Wang ◽  
Scott Lovell ◽  
Kevin P. Battaile ◽  
...  
Keyword(s):  
Binding Site ◽  
Iron Homeostasis ◽  
Anion Binding ◽  
New Class

scholarly journals In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

2005 ◽  
Vol 187 (22) ◽  
pp. 7703-7715 ◽  
Author(s):  
Isabel Delany ◽  
Raffaele Ieva ◽  
Alice Soragni ◽  
Markus Hilleringmann ◽  
Rino Rappuoli ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Binding Site ◽  
Gene Networks ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Target Sequence ◽  
Gene Promoters ◽  
Direct Role ◽  
H Pylori

ABSTRACT Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

scholarly journals The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation.

1992 ◽  
Vol 100 (2) ◽  
pp. 301-339 ◽  
Author(s):  
P J Bjerrum
Keyword(s):  
Ionic Strength ◽  
Binding Site ◽  
Transport System ◽  
Human Erythrocyte ◽  
Binding Constant ◽  
Anion Transport ◽  
High Ionic Strength ◽  
Anion Binding ◽  
Asymmetry Factor ◽  
Sensitive Group

Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate-sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding constant of approximately 38 mM in the alkaline state.

scholarly journals Mouse Bestrophin-2 Is a Bona fide Cl− Channel

2004 ◽  
Vol 123 (4) ◽  
pp. 327-340 ◽  
Author(s):  
Zhiqiang Qu ◽  
Rodolphe Fischmeister ◽  
Criss Hartzell
Keyword(s):  
Binding Site ◽  
Cell Lines ◽  
Electrostatic Interactions ◽  
Anion Binding ◽  
Wild Type ◽  
Cl Channel ◽  
Mammalian Cell Lines ◽  
Conduction Pathway ◽  
Bona Fide ◽  
Cl Conductance

Bestrophins have recently been proposed to comprise a new family of Cl− channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl− channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca2+-activated Cl− current in all three cell lines (EC50 for Ca2+ = 230 nM). The permeability sequence was SCN−: I−: Br−: Cl−: F− (8.2: 1.9: 1.4: 1: 0.5). Although SCN− was highly permeant, its conductance was ∼10% that of Cl− and SCN− blocked Cl− conductance (IC50 = 12 mM). Therefore, SCN− entered the pore more easily than Cl−, but bound more tightly than Cl−. Mutations in S79 altered the relative permeability and conductance for SCN− as expected if S79 contributed to an anion binding site in the channel. PSCN/PCl = 8.2 ± 1.3 for wild-type and 3.9 ± 0.4 for S79C. GSCN/GCl = 0.14 ± 0.03 for wild-type and 0.94 ± 0.04 for S79C. In the S79 mutants, SCN− did not block Cl− conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN−. Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET+ or MTSES− increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl− conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.

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