Anion binding characteristics of the band 3 / 4,4'-dibenzamidostilbene-2,2'-disulfonate binary complex: Evidence for both steric and allosteric interactions

1999 ◽  
Vol 77 (6) ◽  
pp. 543-549 ◽  
Author(s):  
James M Salhany
Keyword(s):  
Steric Hindrance ◽  
Band 3 ◽  
Anion Binding ◽  
Binary Complex ◽  
Allosteric Site ◽  
Ionic Radii ◽  
Affinity Constants ◽  
Access Channel ◽  
Correlation Line ◽  
Transport Site

A novel kinetic approach was used to measure monovalent anion binding to better define the mechanistic basis for competition between stilbenedisulfonates and transportable anions on band 3. An anion-induced acceleration in the release of 4,4prime-dibenzamidostilbene-2,2prime-disulfonate (DBDS) from its complex with band 3 was measured using monovalent anions of various size and relative affinity for the transport site. The K1/2 values for anion binding were determined and correlated with transport site affinity constants obtained from the literature and the dehydrated radius of each anion. The results show that anions with ionic radii of 120-200 pm fall on a well-defined correlation line where the ranking of the K1/2 values matched the ranking of the transport site affinity constants (thiocyanate < nitrate equivalent to bromide < chloride < fluoride). The K1/2 values for the anions on this line were about 4-fold larger than expected for anion binding to inhibitor-free band 3. Such a lowered affinity can be explained in terms of allosteric site-site interactions, since the K1/2 values decreased with increasing anionic size. In contrast, iodide, with an ionic radius of about 212 pm, had a 10-fold lower affinity than predicted by the correlation line established by the smaller monovalent anions. These results indicate that smaller monovalent anions have unobstructed access to the transport site within the band 3 / DBDS binary complex, while iodide experiences significant steric hindrance when binding. The observation of steric hindrance in iodide binding to the band 3 / DBDS binary complex, but not in the binding of smaller monovalent anions, suggests that the stilbenedisulfonate binding site is located at the outer surface of an access channel leading to the transport site.Key words: band 3, anion transport, membrane protein structure, red cell membrane.

scholarly journals Identification and characterization of a second 4,4′-dibenzamido-2,2′-stilbenedisulphonate (DBDS)-binding site on band 3 and its relationship with the anion/proton co-transport function

2005 ◽  
Vol 388 (1) ◽  
pp. 343-353 ◽  
Author(s):  
James M. SALHANY ◽  
Karen S. CORDES ◽  
Renee L. SLOAN
Keyword(s):  
Binding Site ◽  
Proton Transport ◽  
Chloride Concentration ◽  
Band 3 ◽  
Low Ph ◽  
Binding Mechanism ◽  
Access Channel ◽  
Binding Kinetic ◽  
Transport Site

Band 3 mediates both electroneutral AE (anion exchange) and APCT (anion/proton co-transport). Protons activate APCT and inhibit AE with the same pK (∼5.0). SDs (stilbenedisulphonates) bind to a primary, high-affinity site on band 3 and inhibit both AE and APCT functions. In this study, we present fluorescence and kinetic evidence showing that lowering the pH activates a second site on band 3, which binds DBDS (4,4′-dibenzamido-2,2′-stilbenedisulphonate) independently of chloride concentration, and that DBDS binding to the second site inhibits the APCT function of band 3. Activation of the second site correlated with loss of chloride binding to the transport site, thus explaining the lack of competition. The kinetics of DBDS binding at the second site could be simulated by a slow-transition, two-state exclusive binding mechanism (R0↔T0+D↔TD↔RD, where D represents DBDS, R0 and T0 represent alternate conformational states at the second DBDS-binding site, and TD and RD are the same two states with ligand DBDS bound), with a calculated overall Kd of 3.9 μM and a T0+D↔TD dissociation constant of 55 nM. DBDS binding to the primary SD site inhibited approx. 94% of the proton transport at low pH (KI=68.5±11.8 nM). DBDS binding to the second site inhibited approx. 68% of the proton transport (KI=7.27±1.27 μM) in a band 3 construct with all primary SD sites blocked through selective cross-linking by bis(sulphosuccinimidyl)suberate. DBDS inhibition of proton transport at the second site could be simulated quantitatively within the context of the slow-transition, two-state exclusive binding mechanism. We conclude that band 3 contains two DBDS-binding sites that can be occupied simultaneously at low pH. The binding kinetic and transport inhibition characteristics of DBDS interaction with the second site suggest that it may be located within a gated access channel leading to the transport site.

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3

1999 ◽  
Vol 277 (4) ◽  
pp. C791-C799 ◽  
Author(s):  
Sonya Bahar ◽  
Christopher T. Gunter ◽  
Cheryl Wu ◽  
Scott D. Kennedy ◽  
Philip A. Knauf
Keyword(s):  
Binding Site ◽  
External Surface ◽  
Band 3 ◽  
Low Ph ◽  
Anion Binding ◽  
Band 3 Protein ◽  
Proton Binding ◽  
Monovalent Anion ◽  
Woodward’S Reagent K ◽  
Transport Site

Although its primary function is monovalent anion exchange, the band 3 protein also cotransports divalent anions together with protons at low pH. The putative proton binding site, Glu-681 in human erythrocyte band 3, is conserved throughout the anion exchanger family (AE family). To determine whether or not the monovalent anion binding site is located near Glu-681, we modified this residue with Woodward’s reagent K ( N-ethyl-5-phenylisoxazolium-3′-sulfonate; WRK). Measurements of Cl− binding by35Cl-NMR show that external Cl− binds to band 3 even when Cl− transport is inhibited ∼95% by WRK modification of Glu-681. This indicates that the external Cl− binding site is not located near Glu-681 and thus presumably is distant from the proton binding site. DIDS inhibits Cl− binding even when WRK is bound to Glu-681, indicating that the DIDS binding site is also distant from Glu-681. Our data suggest that the DIDS site and probably also the externally facing Cl−transport site are located nearer to the external surface of the membrane than Glu-681.

Molecular design to mimic the copper(II) transport site of human albumin: studies of equilibria between copper(II) and glycylglycyl-L-histidine-N-methyl amide and comparison with human albumin

1976 ◽  
Vol 54 (8) ◽  
pp. 1300-1308 ◽  
Author(s):  
Theo P. A. Kruck ◽  
Show-Jy Lau ◽  
Bibudhendra Sarkar
Keyword(s):  
Ternary System ◽  
Equilibrium Dialysis ◽  
Human Albumin ◽  
Binary Complex ◽  
Mixed Complex ◽  
Free Peptide ◽  
System L ◽  
Peptide Derivative ◽  
Carboxyl Terminal ◽  
Transport Site

In continuing the investigation of designing the specific Cu(II)-transport site of human serum albumin, the peptide derivative glycylglycyl-L-histidine-N-methyl amide was designed to approximate more closely to the native protein. This peptide derivative was synthesized in good yield. The equilibria involved in the binary system, Cu(II)–glycylglycyl-L-histidine-N-methyl amide, have been studied, as well as those in the ternary system, L-histidine–Cu(II)–glycylglycyl-L-histidine-N-methyl amide. This peptide derivative was found to bind Cu(II) exclusively as a 1:1 complex in the pH range 4 to 11, having the same ligand atoms as those for the carboxyl-terminal free peptide and human albumin. However, it was found that glycylglycyl-L-histidine-N-methyl amide bound Cu(II) more strongly than did glycylglycyl-L-histidine, the stability constants being log β1–21 = −0.479 and −1.99 respectively. In the ternary system, only 10% of the mixed complex was detected at pH 7, in comparison to 80% found in the case of the carboxyl-terminal free peptide. This finding agrees well with the increased stability of this peptide binary complex. These observations are also consistent with the results obtained from the equilibrium dialysis experiments. The Cu(II) – peptide amide complex has a dissociation constant of 2.07 × 10−17, indicating a higher binding strength of this peptide derivative for Cu(II) over the native albumin by a factor of 3.

scholarly journals Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

1996 ◽  
Vol 107 (2) ◽  
pp. 271-291 ◽  
Author(s):  
S Q Liu ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Rate Constant ◽  
Band 3 ◽  
Complex Model ◽  
Amino Acid Residues ◽  
Ping Pong ◽  
Extracellular Transport ◽  
Charged Form ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK &gt; 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

The red cell band 3 protein: its role in anion transport

1982 ◽  
Vol 299 (1097) ◽  
pp. 497-507 ◽  
Keyword(s):  
Binding Sites ◽  
Band 3 ◽  
Anion Transport ◽  
Anion Binding ◽  
Hydrophobic Residues ◽  
Ping Pong ◽  
Hydrophilic Residues ◽  
Aqueous Core ◽  
Positively Charged

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60 % of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

Interactions between transport inhibitors at the anion binding sites of the band 3 dimer

Biochemistry ◽  
1981 ◽  
Vol 20 (18) ◽  
pp. 5095-5105 ◽  
Author(s):  
Ian G. Macara ◽  
Lewis C. Cantley
Keyword(s):  
Binding Sites ◽  
Band 3 ◽  
Anion Binding ◽  
Transport Inhibitors

Inhibition of chloride binding to the anion transport site by diethylpyrocarbonate modification of band 3

1990 ◽  
Vol 116 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Naotaka Hamasaki ◽  
Kenji Izuhara ◽  
Kenshi Okubo ◽  
Yoko Kanazawa ◽  
Akira Omachi ◽  
...  
Keyword(s):  
Band 3 ◽  
Anion Transport ◽  
Chloride Binding ◽  
Transport Site
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