Localization of a site of intermolecular cross-linking in human red blood cell band 3 protein.
Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit. In contrast, BSSS forms both intramolecular and intermolecular cross-links between complementary chymotryptic fragments. No intermolecular cross-links between two 35,000-dalton or two 60,000-dalton fragments are detectable. We have localized one end of the BSSS intermolecular cross-link to within 4 residues of the exofacial chymotrypsin cleavage site. The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emerge at the cell surface near the site of intermolecular cross-linking. This is the first detailed information available on the regions of the band 3 primary structure near the interface between subunits.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis*; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013267:Stilbenes / Q000494:pharmacology; D013388:Succinimides / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013267:Stilbenes; D013388:Succinimides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C018564:dihydro-DIDS; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; C035760:bis(sulfosuccinimidyl)suberate; D002918:Chymotrypsin; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid