Oligomerization of a peptide derived from the transmembrane region of the sodium pump gamma subunit: effect of the pathological mutation G41R.
The Na,K-ATPase, or sodium pump, is a ubiquitously expressed membrane-bound enzyme that controls the transmembrane (TM) gradients of sodium and potassium ions in animal cells. The enzyme comprises two subunits, alpha and beta, and in the kidney, is also associated with a small single-spanning membrane protein, the gamma subunit. This 65 amino acid residues protein has been linked to a form of dominant renal hypomagnesaemia resulting from substitution of a highly conserved glycine residue (Gly41) to arginine residue. In order to characterize the quaternary structure of the gamma subunit, and effects of the G41R mutation thereupon, we synthesized a series of peptides (wild-type and mutant) that span the gamma subunit TM region. Using circular dichroism spectroscopy, we show that the 32-amino acid residue peptides are random coils in aqueous buffer but spontaneously adopt an alpha-helical conformation in the presence of detergent micelles (sodium dodecyl sulfate, SDS, and perfluorooctanoate, PFO). Furthermore, fluorescence resonance energy transfer experiments, combined with polyacrylamide gel electrophoresis, demonstrate that while gamma-TM does not self-associate in SDS, it forms oligomers in PFO, a detergent that tolerates relatively weak associations between membrane proteins. Importantly, oligomerization of gamma-TM is abrogated in a peptide that contains either the disease-causing mutation G41R, or the more conservative mutation G41L. On the other hand, a peptide that contains a Gly-to-Arg substitution on a different face of the helix, at position 35, retains its ability to oligomerize. Our results provide evidence for a link between renal hypomagnesaemia and gamma subunit oligomerization.
Therien
Alex G
AG
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, M5G 1X8, Toronto, Ontario, Canada.
Deber
Charles M
CM
D002462:Cell Membrane; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000235:genetics*; D006801:Humans; D007674:Kidney Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics*
D008565:Membrane Proteins; D010446:Peptide Fragments; C416502:FXYD2 protein, human; D000254:Sodium-Potassium-Exchanging ATPase; D005998:Glycine