Therien Alex G, Deber Charles M
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, M5G 1X8, Toronto, Ontario, Canada.
J Mol Biol. 2002 Sep 20;322(3):583-50. doi: 10.1016/s0022-2836(02)00781-7.
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.
钠钾-ATP酶，即钠泵，是一种广泛表达的膜结合酶，可控制动物细胞中钠离子和钾离子的跨膜（TM）梯度。该酶由α和β两个亚基组成，在肾脏中还与一种小的单跨膜蛋白γ亚基相关联。这种由65个氨基酸残基组成的蛋白质与一种显性肾性低镁血症有关，该病症是由一个高度保守的甘氨酸残基（Gly41）被精氨酸残基取代所致。为了表征γ亚基的四级结构以及G41R突变对其的影响，我们合成了一系列跨越γ亚基TM区域的肽段（野生型和突变型）。利用圆二色光谱法，我们发现32个氨基酸残基的肽段在水性缓冲液中呈无规卷曲，但在存在去污剂胶束（十二烷基硫酸钠，SDS，和全氟辛酸，PFO）的情况下会自发形成α-螺旋构象。此外，荧光共振能量转移实验与聚丙烯酰胺凝胶电泳相结合表明，虽然γ-TM在SDS中不会自我缔合，但在PFO中会形成寡聚体，PFO是一种能容忍膜蛋白之间相对较弱缔合的去污剂。重要的是，在含有致病突变G4IR或更保守的突变G41L的肽段中，γ-TM的寡聚化被消除。另一方面，在螺旋不同面上第35位含有甘氨酸到精氨酸取代的肽段仍保留其寡聚化能力。我们的结果为肾性低镁血症与γ亚基寡聚化之间的联系提供了证据。
