Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.     

The carboxyl side chain of glutamate 681 interacts with a chloride binding modifier site that allosterically modulates the dimeric conformational state of band 3 (AE1). Implications for the mechanism of anion/proton cotransport

Glutamate 681 is thought to be located within the transport channel of band 3 (AE1, the chloride/bicarbonate exchanger), where it acts as a proton donor for the anion/proton cotransport function. Here we show that neutralization of the negative charge on glutamate 681 by chemically modifying band 3 with Woodward's reagent K plus sodium borohydride (i.e., the modification process) exposes a cryptic, conformationally active chloride-binding site which functions to modulate allosterically the conformational state of the band 3 dimer. Chloride binding was determined by measuring the effect of increasing chloride concentration on the rate of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) release from band 3 using a stopped-flow fluorescence kinetic inhibitor replacement assay with DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) as the replacing inhibitor. The time course for DBDS release from unmodified, control band 3 was monophasic and exponential. Chloride binding to the transport site accelerated the rate of DBDS release, with the observed rate constant showing a hyperbolic dependence on chloride concentration, while the total change in reaction fluorescence remained constant. After modification of glutamate 681, DBDS release was monophasic in the absence of chloride, but the rapid addition of chloride at constant ionic strength induced a doubling in the fluorescence quantum yield for the bound DBDS molecules. This was associated with the development of 50:50 biphasic kinetics for DBDS release. Such changes were independent of the degree of modification of the band 3 subunit population between the 66% and 91% levels. Titration of the increase in total reaction fluorescence gave an apparent chloride binding K(d) of between 7 and 10 mM, which is 25-40-fold higher in affinity than chloride binding to the transport site. The dependence of the kinetic constants for both phases of the DBDS release reaction on chloride concentration was nonhyperbolic, which contrasts with unmodified band 3, and is indicative of the presence of two classes of chloride-binding sites on the modified transporter. We have also found that the fraction of subunits capable of binding DBDS reversibly, or DIDS covalently, decreased nonlinearly in the absence of chloride as the level of modification of the band 3 subunit population increased. In contrast, the same DBDS binding correlation plot showed a maximum in the presence of saturating chloride. The observation of such nonlinear correlation plots is consistent with a noncooperative dimer model for the modification process, where each dimeric species must possess different properties with respect to stilbenedisulfonate binding capacity and with respect to the spectral-kinetic response of bound stilbenedisulfonate molecules to the addition of chloride. Within the context of this model, the fractions of the three molecular dimeric species (i.e., the unmodified dimer, the dimer with one subunit modified, and the fully modified band 3 dimer) are calculated as a function of the level of modification of the band 3 subunit population. Nonlinear correlation plots are generated by then assigning the following specific properties to each dimeric species. The unmodified dimer binds DBDS but does not change its fluorescence quantum yield upon addition of chloride. The half-modified dimer binds DBDS on both modified and unmodified subunits, and both of those DBDS molecules increase their fluorescence quantum yield by 2-fold when chloride is added, and the system develops 50:50 biphasic DBDS release kinetics. Finally, the model requires that the fully modified dimer does not bind DBDS or DIDS. This model generates theoretical correlation plots that can represent the data presented in this study. We propose that neutralization of glutamate 681 on the half-modified band 3 dimer exposes an allosteric, chloride-binding modifier site which functions to facilitate the anion/proton cotransport process (a) by blocking the "redocking" of the carboxyl side chain of glutamate (thus raising its pK) and (b) by inducing amate (thus raising its pK) and (b) by inducing a conformational change in the band 3 dimer from a symmetrical to an asymmetrical state.     

Inhibition of inorganic anion transport across the human red blood cell membrane by chloride-dependent association of dipyridamole with a stilbene disulfonate binding site on the band 3 protein

The inhibition of inorganic anion transport by dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d] pyrimidine) takes place only in the presence of Cl-, other halides, nitrate or bicarbonate. At any given dipyridamole concentration, the anion flux relative to the flux in the absence of dipyridamole follows the equation: Jrel = (1 + alpha 2[Cl-])/(1 + alpha 4[Cl-]) where alpha 2 and alpha 4 are independent of [Cl-] but dependent on dipyridamole concentration. At high [Cl-] the flux approaches alpha 2/alpha 4, which decreases with increasing dipyridamole concentration. Even when both [Cl-] and dipyridamole concentration assume large values, a small residual flux remains. The equation can be deduced on the assumption that Cl- binding allosterically increases the affinity for dipyridamole binding to band 3 and that the bound dipyridamole produces a non-competitive inhibition of sulfate transport. The mass-law constants for the binding of Cl- and dipyridamole to their respective-binding sites are about 24 mM and 1.5 microM, respectively (pH 6.9, 26 degrees C). Dipyridamole binding leads to a displacement of 4,4'-dibenzoylstilbene-2,2'-disulfonate (DBDS) from the stilbenedisulfonate binding site of band 3. The effect can be predicted quantitatively on the assumption that the Cl- -promoted dipyridamole binding leads to a competitive replacement of the stilbenedisulfonates. For the calculations, the same mass-law constants for binding of Cl- and dipyridamole can be used that were derived from the kinetic studies on Cl- -promoted anion transport inhibition. The newly described Cl- binding site is highly selective with respect to Cl- and other monovalent anion species. There is little competition with SO4(2-), indicating that Cl- binding involves other than purely electrostative forces. The affinity of the binding site to Cl- does not change over the pH range 6.0-7.5. Dipyridamole binds only in its deprotonated state. Binding of the deprotonated dipyridamole is pH-independent over the same range as Cl- binding.     

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

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 > 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.     

Interaction of thiourea with band 3 in human red cell membranes

Summary Although urea transport across the human red cell membrane has been studied extensively, there is disagreement as to whether urea and water permeate the red cell by the same channel. We have suggested that the red cell anion transport protein, band 3, is responsible for both water and urea transport. Thiourea inhibits urea transport and also modulates the normal inhibition of water transport produced by the sulfhydryl reagent,pCMBS. In view of these interactions, we have looked for independent evidence of interaction between thiourea and band 3. Since the fluorescent stilbene anion transport inhibitor, DBDS, increases its fluorescence by two orders of magnitude when bound to band 3 we have used this fluorescence enhancement to study thiourea/band 3 interactions. Our experiments have shown that there is a thiourea binding site on band 3 and we have determined the kinetic and equilibrium constants describing this interaction. Furthermore,pCMBS has been found to modulate the thiourea/band 3 interaction and we have determined the kinetic and equilibrium constants of the interaction in the presence ofpCMBS. These experiments indicate that there is an operational complex which transmits conformational signals among the thiourea,pCMBS and DBDS sites. This finding is consistent with the view that a single protein or protein complex is responsible for all the red cell transport functions in which urea is involved.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors

The band 3 protein of red cells is a transmembrane ion transport protein that catalyzes the one-for-one exchange of anions across the cell membrane. 35Cl NMR studies of Cl- binding to the transport sites of band 3 show that inhibitors of anion transport can be grouped into three classes: (1) transport site inhibitors (examined in this paper), (2) channel-blocking inhibitors (examined in the second of three papers in this issue), and (3) translocation inhibitors (examined in the third of three papers in this issue). Transport site inhibitors fully or partially reduce the affinity of Cl- for the transport site. The dianion 4,4'-di-nitrostilbene-2,2'-disulfonate (DNDS) and the arginine-specific reagent phenylglyoxal (PG) each completely eliminate the transport site 35Cl NMR line broadening, and each compete with Cl- for binding. These results indicate that DNDS and PG share a common inhibitory mechanism involving occupation of the transport site: one of the DNDS negative charges occupies the site, while PG covalently modifies one or more essential positive charges in the site. In contrast, 35Cl NMR line broadening experiments suggest that 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) leaves the transport site partially intact so that the affinity of Cl- for the site is reduced but not destroyed. This result is consistent with a picture in which DIDS binds near the transport site and partially occupies the site.     

Control of red cell urea and water permeability by sulfhydryl reagents

The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2'-disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5.     

35Cl nuclear magnetic resonance line broadening shows that eosin-5-maleimide does not block the external anion access channel of band 3.

It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.     

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

Although its primary function is monovalent anion exchange, theband 3 protein also cotransports divalent anions together with protonsat low pH. The putative proton binding site, Glu-681 in humanerythrocyte band 3, is conserved throughout the anion exchanger family(AE family). To determine whether or not the monovalent anion bindingsite is located near Glu-681, we modified this residue with Woodward'sreagent K(N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements ofCl binding by³⁵Cl-NMR show that externalCl binds to band 3 evenwhen Cl transport isinhibited ~95% by WRK modification of Glu-681. This indicates thatthe external Cl bindingsite is not located near Glu-681 and thus presumably is distant fromthe proton binding site. DIDS inhibitsCl binding even when WRK isbound to Glu-681, indicating that the DIDS binding site is also distantfrom Glu-681. Our data suggest that the DIDS site and probably also theexternally facing Cltransport site are located nearer to the external surface of themembrane than Glu-681.

     

Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene

Summary The fluorescence enhancement of 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K _d =3.8 m) and rat (3.2 m); 100 m dihydro-4,4-diisothiocyano-2,2-disulfonic stilbene (H₂DIDS) blocked >95% of binding. H₂DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK _d doubled with 10mm SO₄, 50mm HCO₃ and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO₄, HCO₃ and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used³⁵SO₄ uptake to characterize several modes of rabbit BLM anion transport: H/SO₄ and Na/SO₄ cotransport, and Cl/SO₄ countertransport. Each transport process was electroneutral and was inhibited by H₂DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK _t 's for DBDS (3–20 m) were similar toK _d for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene.     

Steric restrictions on the binding of large metal ions to serum transferrin

Apotransferrin in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid at 25 degrees C and pH 7.4 was titrated with acidic solutions of Lu3+, Tb3+, and Eu3+. Metal binding at the two specific metal-binding sites of transferrin was followed from changes in the difference UV spectra at 245 nm. The binding of Tb3+ was also followed from changes in the fluorescence emission spectrum at 549 nm. Apotransferrin was titrated with solutions containing varying ratios of the metal ion and the competitive chelating agent nitrilotriacetic acid, and metal-transferrin binding constants were calculated by nonlinear least-squares fits of the absorbance as a function of titrant added. The sequential carbonate-independent equilibrium constants for the binding of two metal ions are log KM1 = 11.08 and log KM2 = 7.93 for Lu3+, log KM1 = 11.20 and log KM2 = 7.61 for Tb3+, and log KM1 = 9.66 and log KM2 = 7.27 for Eu3+. Titrations of both C-terminal and N-terminal monoferric transferrins indicate that all of these metal ions bind more strongly to the C-terminal binding site. The trend in log K values as a function of the lanthanide ionic radius has been evaluated both by plots of log K versus the metal ion charge/radius ratio and by linear free-energy relationships in which binding constants for complexes of the larger lanthanides are plotted versus the binding constants for complexes with the smallest lanthanide, Lu3+. Both methods indicate that there is a sharp drop in the binding constants for the C-terminal binding site for metals larger than Tb3+. This decrease is attributed to a steric hindrance to the binding of the larger cations. The steric effect is not as strong for metal binding at the N-terminal site. As a result, the selectivity for binding to the C-terminal site, which is quite high for the smaller lanthanides, drops sharply on going from Tb3+ to Nd3+.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Temperature dependence of anion transport inhibitor binding to human red cell membranes

The binding characteristics of the inhibitor of anion transport in human red cells, 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS), to the anion transport protein of red cell ghost membranes in buffer containing 150 mM NaCl have been measured over the temperature range 0-30 degrees C by equilibrium and stopped-flow fluorescence methods. The equilibrium dissociation constant Keq, increased with temperature. No evidence of a 'break' in the ln(Keq) vs. 1/T plot was found. The standard dissociation enthalpy and entropy changes calculated from the temperature dependence are 9.1 +/- 0.9 kcal/mol and 3.2 +/- 0.3 e.u., respectively. Stopped-flow kinetic studies resolve the overall binding into two steps: a bimolecular association of DBDS with the anion transport protein, followed by a unimolecular rearrangement of the DBDS-protein complex. The rate constants for the individual steps in the binding mechanism can be determined from an analysis of the concentration dependence of the binding time course. Arrhenius plots of the rate constants showed no evidence of a break. Activation energies for the individual steps in the binding mechanism are 11.6 +/- 0.9 kcal/mol (bimolecular, forward step), 17 +/- 2 kcal/mol (bimolecular, reverse step), 6.4 +/- 2.3 kcal/mol (unimolecular, forward step), and 10.6 +/- 1.9 kcal/mol (unimolecular, reverse step). Our results indicate that there is an appreciable enthalpic energy barrier for the bimolecular association of DBDS with the transport protein, and appreciable enthalpic and entropic barriers for the unimolecular rearrangement of the DBDS-protein complex.     

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Fr?lich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.     

Anion binding properties of human serum albumin from halide ion quadrupole relaxation.

The nuclear magnetic quadrupole relaxation enhancement of 35Cl-, 81Br-, and 12I- anions on binding to human serum albumin has been studied under conditions of variable protein and anion concentration and also in the presence of simple inorganic, amphiphilic, and complex anions which compete with the halide ions for the protein anion binding sites. Two classes of anion binding sites with greatly different binding constans were identified. Experiments at variable halide ion concentration were employed to determin the Cl- and I- binding constants. By means of 35 Cl nuclear magnetic resonance (NMR) the relative affinity for different anions was determined by competition experiments for both the strong and the weak anion binding sites. Anion binding follows the sequence SO42- smaller than F- smaller than CH3COO- smaller than Ci- smaller Br- smaller than NO3- smaller than I- smaller than ClO4- smaller than SCN- smaller than Pt(CN)42- smaller than Au(CN)2- smaller than CH3(CH2)11OSO3- for the high affinity sites, and the sequence SO42- congruent to F- congruent to Cl- smaller CH3COO- smaller than NO3- smaller than Br- smaller than I- smaller than ClO4- smaller than SCN- for the low affinity sites. These series are nearly identical with the well-known lyotropic series. Consequently, those effects of anions on proteins described by the lyotropic series can be correlated with the affinities of the anions for binding to the protein. The data suggest that the physical nature of the interaction is the same for both types of biding sites, and that the differences in affinity between different binding sites must be explained in terms of tertiary structure. Analogous experiments performed using 127I- quadrupole relaxation gave results very similar to those obtained with 35Cl-. A comparison between the Cl-, Br- and I- ions revealed that, as a result of the increasing affinity for the weak anion binding sites in the series Cl- smaller than Br- smaller than I-, Cl- is much more useful as a probe for the specific anion binding sites than the other two halide ions. The findings with human serum albumin in this and other respects are probably of general relevance in studies of protein-anion interactions. In addition to competition experiments, the magnitude of the relaxation rate is also discussed. Line broadening not related to anion binding to the protein is found to be small. A comparison of transverse and longitudinal 35Cl relaxation rates gives a value for the quadrupole coupling constant of the high affinity sites in good agreement with a calculated coupling constant assuming anion binding to arginine.     

The influence of Mg2+ on anion binding to sarcoplasmic reticulum membranes as detected by 35Cl-NMR.

35Cl-NMR spectroscopy has been used to study the competition between anions, including nucleotides, on skeletal muscle sarcoplasmic reticulum membranes. Different chloride binding sites can be distinguished according to their Mg2+ sensitivity. Phosphate binding is enhanced by Mg2+ whereas the anion transport inhibitor pyridoxalphosphate-6-azophenyl-2'-sulfonic acid (PPAPS) binding is not. The affinity of the enzyme for the Mg-adenylyl imidodiphosphate (MgAMP-PNP) complex is decreased whereas that for MgATP is increased. Three sets of binding sites can be discriminated from which chloride is displaced by different anions with varying efficiency. High affinity binding of AMP-PNP and PPAPS occurs at the same site, that can also be occupied by phosphate. Low-affinity binding of PPAPS and AMP-PNP also coincides, but in a site where phosphate binding is negligible. ATP and ADP bind to both sites. In the presence of Mg2+ a third anion binding site can be occupied by phosphate but neither by AMP-PNP nor PPAPS.     

Kinetic dependence to HIV-1 entry inhibition

Infection by human immunodeficiency virus type 1 (HIV-1) involves the fusion of viral and cellular membranes mediated by formation of the gp41 trimer-of-hairpins. A designed protein, 5-Helix, targets the C-terminal region of the gp41 ectodomain, disrupting trimer-of-hairpins formation and blocking viral entry. Here we show that the nanomolar inhibitory potency of 5-Helix (IC50 approximately 6 nm) is 4 orders of magnitude larger than its subpicomolar binding affinity (K(D) approximately 0.6 pm). This discrepancy results from the transient exposure of the 5-Helix binding site on gp41. As a consequence, inhibitory potency is determined by the association rate, not by binding affinity. For a series of 5-Helix variants with mutations in their gp41 binding sites, the IC50 and K(D) values poorly correlate. By contrast, an inverse relationship between IC50 values and association rate constants (k(on)) extends for over 2 orders of magnitude. The kinetic dependence to inhibition places temporal restrictions on an intermediate state of HIV-1 membrane fusion and suggests that access to the C-terminal region of the gp41 ectodomain is largely free from steric hindrance. Our results support the importance of association kinetics in the development of improved HIV-1 fusion inhibitors.     

Conformational changes in human red cell membrane proteins induced by sugar binding

Summary We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A.L., Carruthers, A., (1987)J. Biol. Chem. 262:5464–5475] to displace cytochalasin B (CB), the specificd-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75mm maltose, a nontransportable disaccharide which also displaces CB, caused a timedependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (±DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 m DBDS increased the damping term activation energy, 9.5±2.8 kcal mol^–1 deg^–1, by a factor of four to 39.7±5.1 kcal mol^–1 deg^–1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.