Studies on the structure of a transmembrane region and a cytoplasmic loop of the human red cell anion exchanger (band 3, AE1).

Band 3 (AEl), the major human red cell integral protein, is a bifunctional polytopic protein containing 911 amino acids [l]. The protein contains two structurally distinct domains which have different functions: the N-terminal 40 kDa domain (residues 1-360) is located within the red cell cytoplasm and acts as an anchorage point for the red cell skeleton, and also binds several glycolytic enzymes [Z, 31; the membrane-associated 55 kDa C-terminal domain (residues 361-91 1) carries out the obligatory exchange transport of anions [4]. There have been several recent reviews of the protein [S-81. The combination of hydropathy analysis, proteolytic cleavage and chemical modification studies suggested that the membrane domain of band 3 contained up to 14 membrane spans [6,9], but a more recent study indicates that the polypeptide may only span the membrane 12 times [lo]. CD studies have shown that the membrane domain of band 3 is substantially helical (60%), with the transmembrane segments being almost entirely helical [ 111. NMR of synthetic peptides representing the first and second transmembrane segments of band 3 suggest that these are also predominantly helical in organic solvents [12]. It is likely that the structure of band 3, like other polytopic membrane proteins which are mainly helical, is based on a tightly packed helical bundle with the loops that connect the helical spans extending into the aqueous environment at the membrane surface. Although band 3 is abundant in red cells and relatively simple to purify, it has been difficult to obtain crystals which diffract to high resolution. The two-dimensional crystals that have been obtained have thus far only yielded low resolution (2OA) structures [13-151. The membrane domain of band 3 is too large to be studied directly by solution NMR. An approach we are taking to obtain structural infor-