Characterization of the stilbenedisulphonate binding site on band 3 Memphis variant II (Pro-854-->Leu).
Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56-->Glu within the cytoplasmic domain, and Pro-854-->Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4'-di-isothiocyanatodihydro-2, 2'-stilbenedisulphonate) to 'lysine A' (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS 'off' rate constant, but no difference was found between mutant and control when DBDS (4,4'-dibenzamido-2,2'-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) 'off' rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4'-di-isothiocyanato-2,2'-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854-->Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.
Salhany
J M
JM
Veterans Administration Medical Center, Omaha, NE, USA.
Sloan
R L
RL
Schopfer
L M
LM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000737:chemistry / Q000378:metabolism*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D001457:Anion Exchange Protein 1, Erythrocyte; D003432:Cross-Linking Reagents; C100810:band 3 protein Memphis variant II; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid