Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals

Because nearly all structure/function studies on Na ؉ / K ؉ -ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting ␣,␣-, ␣,␤-, ␤,␤-, and ␣,␥-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation. This solubilized enzyme had no ATPase activity and was not phosphorylated by ATP, but it retained the ability to occlude Rb ؉ and Na ؉ . This, and comparison of cross-linking patterns obtained with different reagents, suggested that the transmembrane domains of the enzyme are more resistant to SDSinduced unfolding than its other domains. These findings (a) indicate that the partially unfolded oligomer(s) retaining partial function is the intermediate in the SDS-induced denaturation of the native membrane enzyme having the minimum oligomeric structure of (␣,␤,␥) 2 and (b) suggest potential functions for Na ؉ /K ؉ -ATPase with intrinsically unfolded domains. Mixtures of solubilized/ partially unfolded enzyme and membrane-bound enzyme exhibited cross-linking patterns and Na ؉ occlusion capacities different from those of either enzyme species, suggesting that the two interact. Formation of the partially unfolded enzyme during standard purification procedure for the preparation of the membrane-bound enzyme was shown, indicating that it is necessary to ensure the separation of the partially unfolded enzyme from the membrane-bound enzyme to avoid the distortion of the properties of the latter.

Section: Table Imentioning

confidence: 59%

“…The kidney enzyme preparation used here contains a third subunit, ␥ (ϳ7 kDa), that regulates its function (3,4,36,37,48). To see whether ␥ interaction with the other subunits could be detected by cross-linking in the native and the TDS-solubilized enzymes, experiments of Fig.…”

Section: Sds-and Tds-solubilized Enzymes Retain Quaternary Structure-mentioning

confidence: 99%

Interaction of SDS with Na+/K+-ATPase

Ivanov

Gable

Askari

2004

“…Thus, sub-critical micelle concentrations of detergent that impair the protein-protein hydrophobic interactions relieved PLMS inhibition of Na,K-ATPase (21). Previous investigations have suggested ␥ to be localized close to the C terminus of the ␣-subunit (27,28). Compared with the three-dimensional structure determination of SERCA (68), this position would also be close to the N terminus/M1-M2 transmembrane segments of the Na,K-ATPase ␣-subunit, i.e.…”

Section: Discussionmentioning

confidence: 98%

“…Spatial localization of the ␥-subunit has been indirectly inferred from cryo-electron microscopy of two-dimensional crystals (27) or from thermal denaturation experiments (28). They seem to indicate that the ␥-subunit is associated with the C terminus of the ␣-subunit being located either between the M2/M9 or the M9/M10 transmembrane segments.…”

mentioning

confidence: 99%

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

Mahmmoud

Cramb

Maunsbach

et al. 2003

Self Cite

In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase ␣-subunit and PLMS showed the presence of ␣ and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E 1 ϳP 3 E 2 -P reaction, which is partially rate-limiting in shark, and the K ؉ deocclusion reaction, E 2 (K) 3 E 1 , are accelerated. The latter may explain the finding that the apparent Na ؉ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase ␣-subunit is important for the modulation of shark Na,K-ATPase activity.

“…Experiments based on thermal denaturation suggest that the association of FXYD2 occurs with transmembrane (TM) 1 domains 8 -10 (18). Moreover, a recent model (19), deduced from an electron crystallographic analysis at 9.5-Å resolution of renal Na,K-ATPase and by taking as a basis the high resolution structure of the Ca-ATPase (20), predicts that FXYD2 is located in a pocket made up of TM9, TM6, TM4, and TM2 of the Na,K-ATPase ␣ subunit. In this study, we investigated the role of TM9 of the Na,K-ATPase ␣ subunit in the structural and functional interaction with FXYD proteins.…”

mentioning

confidence: 99%

Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins

Grosdidier

Crambert

et al. 2004

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K ؉ and Na ؉ affinity. Little is known about the interaction sites between the Na,KATPase ␣ subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase ␣ subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe 956 , Glu 960 , Leu 964 , and Phe 967 in TM9 of the Na,K-ATPase ␣ subunit represent one face interacting with the three FXYD proteins. Leu 964 and Phe 967 contribute to the efficient association of FXYD proteins with the Na,K-ATPase ␣ subunit, whereas Phe 956 and Glu 960 are essential for the transmission of the functional effect of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase. The relative contribution of Phe 956 and Glu 960 to the K ؉ effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase. In contrast to the effect on the apparent K ؉ affinity, Phe 956 and Glu 960 are not involved in the effect of FXYD2 and FXYD4 on the apparent Na ؉ affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/ FXYD7 complex, which also predicts the importance of Phe 956 , Glu 960 , Leu 964 , and Phe 967 in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase ␣ subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K ؉ affinity of Na,K-ATPase.