Structural studies on the effects of the deletion in the red cell anion exchanger (band 3, AE1) associated with South East Asian ovalocytosis.
We have carried out a solution-state NMR study of synthetic peptides patterned on the first membrane span of normal human band 3, and the same region of the mutant band 3 present in Southeast Asian ovalocytosis (SAO) which has a nine amino acid deletion. In 1:1 (v/v) chloroform/methanol, the 42 residue normal peptide (R389-K430) consisted of three helical regions. The slow solvent exchange of backbone amide protons revealed the helix from P403 to A416 was more stable than the "cytoplasmic" N-terminal helix from P391 to A400. These helices were separated by a sharp bend at P403, which is probably located at the boundary between the cytoplasmic domain and the first transmembrane span. The SAO deletion (A400-A408) removed the bend at P403, to leave a stable helix from P391 to A416 containing the residuum of the normal first transmembrane helix and with a hydrophobic turn replaced by a polar turn in the SAO peptide. Insertion of fragments of normal band 3 and band 3 SAO into microsomal membranes was investigated using a cell free translation system. A fragment composed of the cytoplasmic domain and the putative first membrane domain of normal band 3 (B3(1)) inserted stably into the membrane. However, the corresponding fragment of band 3 SAO [SAO(1)] did not integrate stably into membranes. Our results suggest that in SAO band 3, the region of the first membrane span of normal band 3 does not integrate properly into the membrane because it lacks a sufficiently long hydrophobic segment, and the deletion also disrupts a conserved structural subdomain at the membrane surface.
Chambers
E J
EJ
Department of Biochemistry School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
Bloomberg
G B
GB
Ring
S M
SM
Tanner
M J
MJ
D000577:Amides / Q000378:metabolism; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics*; D001208:Asia; D004612:Elliptocytosis, Hereditary / Q000235:genetics*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D014176:Protein Biosynthesis / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics
D000577:Amides; D001457:Anion Exchange Protein 1, Erythrocyte; D008565:Membrane Proteins; D010446:Peptide Fragments; D011392:Proline