Kinetic Evidence for Ternary Complex Formation and Allosteric Interactions in Chloride and Stilbenedisulfonate Binding to Band 3

Abstract

The molecular basis for chloride and stilbenedisulfonate interaction with band 3 was investigated by measuring the kinetics of stilbenedisulfonate release from its complex with the transporter. We found that 150 mM NaCl accelerated the rate of release of DBDS (4,4'-dibenzamidostilbene-2,2'-dibenzamidostilbene-2,2'-disu lfonate) and H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate) by more than 10-fold at constant ionic strength. The acceleration effect saturated as a function of chloride concentration. This is an indication of specific binding within a ternary complex involving stilbenedisulfonate, chloride, and band 3. To see if stilbenedisulfonates block an access channel to the transport site, we studied the effect of rapidly mixing DBDS-saturated resealed ghosts with chloride at constant ionic strength and osmotic pressure. Once again, we observe a large, uniform acceleration in the rate of DBDS release. These findings are not consistent with molecular models where stilbenedisulfonates are proposed to block access to a deeper transport site. We suggest that the intramonomeric stilbenedisulfonate site is not located on the chloride transport pathway but rather interacts with the transport site though heterotropic allosteric site-site interactions. On the basis of our kinetic evidence for ternary complex formation and on transport inhibition evidence in the literature showing a linear dependence of KI-app on substrate, we suggest that stilbenedisulfonates are linear mixed-type inhibitors of band 3 anion exchange, not pure competitive inhibitors as has been assumed on the basis of analysis of transport inhibition data alone.

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

“…At saturation, the slow-phase rate is equal to k2 + k-2 (20), which can be used to calculate k2 by subtracting k-2 from the observed rate. The k-2 value was determined experimentally from the replacement reaction (11,15). The rate constant from the replacement reaction was found to be virtually identical to the value of k-2 determined from a fit of Eq.…”

“…The reactions were all monophasic and exponential (see figure 4 of ref. 15), and there was no significant difference in the DBDS-release rate between wild-type band 3 and band 3 HT (Table 1). Furthermore, chloride accelerated the reaction to the same degree in both cases (Table 1).…”

“…We use the difference in protein fluorescence quenching due to H2DIDS and DIDS reversible binding to band 3 to monitor the kinetics of replacement of H2DIDS by DIDS, as described (11). This replacement reaction offers a direct measure of the rate of H2DIDS release from its reversible complex with band 3 (11,15).…”

“…DBDS/DIDS Replacement Reaction. Since it is technically difficult to measure the DIDS off rate because of the rapidity of the covalent adduct formation reaction (10), we used our DBDS/DIDS replacement reaction as another example of stilbenedisulfonate release from band 3 (15). The reactions were all monophasic and exponential (see figure 4 of ref.…”

Cysteine-directed cross-linking localizes regions of the human erythrocyte anion-exchange protein (AE1) relative to the dimeric interface

“…Although our findings cannot discriminate between an anion channel located at the dimeric interface and one located within each monomer, we are able to refine an allosteric model for anion exchange [15,16]. Previous work indicated the importance of monomer-monomer contacts in anion transport, since perturbation of these contacts following stilbene disulphonate binding may provide an allosteric mechanism for inhibition by these ligands [17,36]. The present findings suggest that interaction between TMs rather than loop regions at the dimeric interface is important for the allosteric model of anion transport, since constraining the conformation\dynamics of extramembraneous regions of the protein located near the dimeric interface by cross-linking did not alter AE1 transport function.…”