Structural characterization of the cytosolic domain of kidney chloride/bicarbonate anion exchanger 1 (kAE1).
Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed in alpha-intercalated cells in the collecting ducts of the kidney where it mediates electroneutral chloride/bicarbonate exchange. Human kAE1 is a truncated form of erythroid AE1 missing the first 65 residues of the N-terminal cytosolic domain, which includes a disordered acidic region (residues 1-54) and the first beta-strand (residues 55-65) of the folded region. Unlike erythroid AE1, kAE1 does not bind deoxyhemoglobin, glycolytic enzymes, or cytoskeletal components. To understand the effect of the N-terminal deletion on the structure of the cytosolic domain, we performed an extensive biophysical analysis on His 6 tagged cytosolic domains of erythroid AE1 (cdAE1), kidney AE1 (cdkAE1), and a novel truncation mutant (cdDelta54AE1) missing the first 54 residues, but retaining the beta-strand. Circular dichroism did not detect any major differences in secondary structure, and sedimentation analyses showed that all three proteins were dimeric. Differential scanning calorimetry revealed that cdAE1 and cdDelta54AE1 had similar thermal stabilities with midpoints of transition higher than cdkAE1. cdAE1 and cdDelta54AE1 underwent similar pH-dependent fluorescence changes, while cdkAE1 exhibited a higher intrinsic fluorescence at neutral and acidic pH. Urea denaturation resulted in dequenching of tryptophan fluorescence in cdAE1, while tryptophans in cdkAE1 were already dequenched in the native state. We conclude that the absence of the central beta-strand in cdkAE1 results in a less stable and more open structure than cdAE1. This structural change, in addition to the loss of the acidic amino-terminal region, may account for the altered protein binding properties of kAE1.
Pang
Allison J
AJ
Department of Biochemistry, University of Toronto, 1 King's College Circle, Medical Sciences Building, Room 5216, Toronto, Ontario M5S 1A8, Canada.
Bustos
Susan P
SP
Reithmeier
Reinhart A F
RA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003600:Cytosol / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D008958:Models, Molecular; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation
D001457:Anion Exchange Protein 1, Erythrocyte; D014364:Tryptophan