Factors determining the conformation and quaternary structure of isolated human erythrocyte band 3 in detergent solution

Abstract

Fluorescence spectroscopy was used to follow the kinetics of covalent binding of DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) to isolated band 3 in C12E8. We have discovered a dilution-induced loss in the ability of band 3 monomer to form a covalent adduct with DIDS. The loss in DIDS reactivity with dilution followed a 50:50 biphasic time course despite the use of a homogeneous preparation of band 3 oligomers. The loss in reactivity generally correlated with the association of band 3 dimers and tetramers to higher oligomeric structures. The final aggregated product was capable of binding BADS (4-benzamido-4'-amino-2,2'-stilbenedisulfonate) reversibly, but with an affinity nearly 30-fold lower than that of the starting material. Removal of the cytoplasmic domain of band 3 slowed the conformational interconversion of the integral domain by about 5-fold and inhibited the aggregation process. The conformational interconversion was slowed in the presence of 150 mM chloride but not in 90 mM sulfate. Covalent binding of DIDS inhibited the aggregation of band 3. Addition of 250 microM lipid inhibited both the loss of DIDS reactivity and the protein aggregation process. While several types of lipid offer protection, phosphatidic acid accelerated the decay process by eliminating the biphasicity. We conclude that the conformation of the integral domain of band 3 can be modulated allosterically by the addition of ligands, including various lipids. The results offer direct evidence for cooperative interactions between band 3 subunits during loss of activity, and they show that the cytoplasmic domain participates in the control of this transition.

Hydrodynamic Properties of Human Erythrocyte Band 3 Solubilized in Reduced Triton X-100

“…In a typical experiment band 3 was purified at a concentration of 4 mg/ml (from either method of preparation) and subsequently diluted at least fourfold before centrifugation. A similar dilution-induced aggregation has been observed by Schopfer and Salhany (1992).…”

“…When the results obtained from this study are considered with the findings of others concerning 1) putative conformational changes associated with band 3 oligomerization (Casey and Reithmeier, 1991;Salhany et al, 1997;Schopfer and Salhany, 1992;Vince et al, 1997); 2) the role of ligands in stabilizing dimeric band 3 in vitro (Maneri and Low, 1989;Schopfer and Salhany, 1992;Vince et al, 1997); and 3) the binding of ankyrin to band 3, which shifts the monomer/dimer/tetramer equilibrium in favor of the tetramer (Huber et al, 1996), a model describing solubilized band 3 hydrodynamic behavior may be proposed: FIGURE 6 Fitting of data obtained from the Omega function analysis. The best fit for the sample at 5000 rpm was obtained from a dimer/ tetramer/hexamer model of the pooled data from Fig.…”

“…It is generally accepted that band 3 exists as a mixture of stable dimers and tetramers in situ. The physiological significance of the observed [monomer/dimer/tetramer] 100 band 3 self-association is therefore uncertain, but this form of the protein has been isolated by others using different purification procedures and seems not to arise from an artifact of the purification procedure described in this study (Casey and Reithmeier, 1991;Schopfer and Salhany, 1992). Arguments that may explain why studies fail to reveal evidence for such selfassociation behavior occurring in situ include the following: 1) ankyrin binds only to tetrameric band 3 and therefore provides a mechanism to drive the monomer/dimer/tetramer self-association toward an exclusively [tetramer-ankyrin] complex (see model); 2) only after removal of ankyrin can the self-associating form of band 3 equilibrate in situ to a mixture of monomers, dimers, and tetramers entirely consistent with the monomer/dimer/tetramer 7 [tetramerankyrin] model presented here (Van Dort et al, 1998); and 3) in situ equilibration of band 3 oligomers occurs very slowly compared to that observed in detergent solution, which may explain why other studies fail to detect the presence of band 3 monomers in situ after removal of ankyrin (Van Dort et al, 1998).…”

“…There are conflicting reports about the oligomeric state of purified band 3 in detergent solution. The protein has been characterized as stable dimers (Clarke, 1975;Pinder et al, 1995), a self-associating mixture of monomers-dimers-tetramers (Pappert and Schubert, 1983;Schubert et al, 1983), and a polydisperse mixture of either monomers and dimers (Lukacovic et al, 1981), dimers and tetramers (Nakashima and Makino, 1980;Nakashima et al, 1981;Casey and Reithmeier, 1991;Schopfer and Salhany, 1992), or dimers and hexamers (Wong, 1993). The different hydrodynamic characteristics associated with different band 3 preparations indicate that the exact experimental conditions under which the protein is purified and examined are critical.…”

“…The concentration of purified band 3 in detergent solution, a parameter that will vary with purification protocol, affects the oligomeric state of the protein (Schopfer and Salhany, 1992). Dilution of solubilized band 3 initiated irreversible aggregation, a process that could be slowed by the addition of substrate anions, stilbene disulfonates, and certain types of phospholipid.…”