Journal of Molecular Biology
Janus Model of The Na,K-ATPase β-Subunit Transmembrane Domain: Distinct Faces Mediate α/β Assembly and β-β Homo-oligomerization
Introduction
Na,K-ATPase is a ubiquitously expressed, plasma membrane-bound enzyme that mediates the ATP-dependent transport of three sodium ions out of the cell and two potassium ions into the cell. The ion transduction function of Na,K-ATPase is critical for the maintenance of ion homeostasis within the cell, and has been well studied.1 Na,K-ATPase is an oligomeric enzyme consisting of two essential subunits, the α-subunit (Na,K-α), which is the catalytic subunit, and a regulatory β-subunit (Na,K-β), which is required for the translation, stability, and membrane insertion of Na,K-α.2,3 Thus, the primary role of Na,K-β is to assist the Na,K-ATPase ion transport function via its interaction with Na,K-α. Recently, we and others have shown that Na,K-β functions also as a cell–cell adhesion molecule.4., 5., 6., 7. The structural features of Na,K-β involved in the regulation of Na,K-ATPase enzyme activity and cell–cell adhesion have not been well characterized.
Na,K-β is a type II transmembrane protein with a short cytoplasmic domain and a large glycosylated extracellular domain. Three different isoforms of Na,K-β are known, β1, β2, and β3. The transmembrane domain (TMD) of β1 (the β isoform used in this study) is highly conserved (99%) throughout the animal kingdom, ranging from human to chicken. Amongst the different isoforms of Na,K-β, there is only a 30–35% identity over the entire protein sequence; however, their TMDs are 57–61% identical. This extensive sequence conservation within the TMD of Na,K-β suggests that Na,K-β TMD must play an important role in the structure and function of Na,K-ATPase.
A type II membrane protein typically spans the membrane as a stable α-helix, a conformation that internally satisfies the backbone hydrogen bonding groups in the hydrophobic lipid environment.8 Folding and assembly of membrane proteins is often supported by specific interactions between the preformed helical transmembrane segments.9., 10., 11., 12., 13. Sequence motifs have been identified in transmembrane helices that are strongly associated with helix–helix interactions.14., 15., 16., 17., 18. Here, we show that the TMD of Na,K-β has both a conserved heptad repeat and a glycine zipper motif on two different faces, suggesting the possibility that Na,K-β could interact with two different partners. To test this hypothesis, we introduced mutations into the two faces of the Na,K-β TMD to disrupt lateral association with its binding partners. We find that one face of the Na,K-β TMD interacts with Na,K-α, and regulates enzyme activity, while the other face is involved in Na,K-β self-association, which is important for the cell–cell adhesion function of this protein. Thus, Na,K-β is a key structural component of the Na,K-ATPase monomer, and it serves to oligomerize the protein.
Section snippets
Packing interface of Na,K-β transmembrane domain
Certain sequence motifs are prevalent in TM helix–helix interactions. One of the best characterized sequence motifs known to mediate helix oligomerization is the GxxxG motif.9,19 As shown in Figure 1(b), the putative TMD of Na,K-β contains two tandem GxxxG sequences (GxxxGxxxG), known also as a glycine zipper.20 Glycine zippers have been shown to drive TM helix oligomerization strongly, hinting at the possibility that the glycine zipper face of Na,K-β could form an interaction surface with
Discussion
In this study, we have identified heptad repeat motif and glycine zipper motif in the TMD of Na,K-β by structure prediction, motif identification and evolutionary analysis. We validated our predictions by mutagenesis of critical residues within the motifs, and by analyzing the mutants in specific functional assays. We demonstrated that the heptad repeat motif and the glycine zipper motif are involved in two different interactions. Na,K-β interacts with Na,K-α via the heptad repeat motif, and
Sequence conservation scoring
The program ConSeq‡, was used to map the sequence conservation reflected in the multiple sequence alignment of canine Na,K-β (Swiss-Prot accession number P06583) with 34 representative sequence homologs.41 This server compares the sequence of a reference protein with the proteins deposited in Swiss-Prot,42 and finds those that are homologous to the sequence. The number of PSI-BLAST iterations and the E-value cutoff used were 1 and 0.001, respectively. All the
Acknowledgements
This work was supported by NIH DK56216. We thank Dr Donald M. Engelman (Yale University, New Haven, CT), for providing us the reagents for the TOXCAT assay, Dr William James Ball, Jr for Na,K-ATPase antibodies and Dr Robert Farley for canine Na,K-β cDNA.
References (44)
- et al.
Modeling transmembrane helical oligomers
Curr. Opin. Struct. Biol.
(1997) - et al.
A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments
J. Biol. Chem.
(1999) - et al.
Motifs of serine and threonine can drive association of transmembrane helices
J. Mol. Biol.
(2002) - et al.
Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions
J. Mol. Biol.
(2000) - et al.
Biosynthesis of the Na,K-ATPase in Madin-Darby canine kidney cells. Activation and cell surface delivery
J. Biol. Chem.
(1990) - et al.
The GxxxG motif: a framework for transmembrane helix-helix association
J. Mol. Biol.
(2000) Cadherins and catenins: interactions and functions in embryonic development
Curr. Opin. Cell Biol.
(1994)Lateral dimerization of the E-cadherin extracellular domain is necessary but not sufficient for adhesive activity
J. Biol. Chem.
(2002)Coiled coils: new structures and new functions
Trends Biochem. Sci.
(1996)- et al.
A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure
J. Biol. Chem.
(1996)
Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning
J. Biol. Chem.
Subunit interactions in the Na,K-ATPase explored with the yeast two-hybrid system
J. Biol. Chem.
Characterization of disulfide cross-links between fragments of proteolyzed Na,K-ATPase. Implications for spatial organization of trans-membrane helices
J. Biol. Chem.
The single transmembrane domains of ErbB receptors self-associate in cell membranes
J. Biol. Chem.
Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes
J. Biol. Chem.
Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits
J. Biol. Chem.
Association of prostate-specific membrane antigen with caveolin-1 and its caveolae-dependent internalization in microvascular endothelial cells: Implications for targeting to tumor vasculature
Microvasc. Res.
Identification of the site of inhibition by omeprazole of a alpha-beta fusion protein of the H,K-ATPase using site-directed mutagenesis
J. Biol. Chem.
Ion movements through the sodium pump
Annu. Rev. Physiol.
Na,K-ATPase beta1-subunit increases the translation efficiency of the alpha1-subunit in MSV-MDCK cells
Mol. Biol. Cell
The functional role of beta subunits in oligomeric P-type ATPases
J. Bioenerg. Biomembr.
Na,K-ATPase beta-subunit is required for epithelial polarization, suppression of invasion, and cell motility
Mol. Biol. Cell
Cited by (47)
Experimental determination and data-driven prediction of homotypic transmembrane domain interfaces
2020, Computational and Structural Biotechnology JournalCitation Excerpt :Based on case studies alone, the presence of these motifs is often assumed to indicate self-interaction or the presence of an interface. As a consequence, motifs are usually the first residues to be targeted in mutagenesis experiments [35–39]. There is a strong need for statistical analyses to objectively define the importance of these motifs and other sequence properties in homotypic TMD interaction.
Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay
2016, Biochimica et Biophysica Acta - BiomembranesMultimeric species in equilibrium in detergent-solubilized Na,K-ATPase
2016, International Journal of Biological MacromoleculesSodium-calcium exchanger 1 regulates epithelial cell migration via calcium-dependent extracellular signal-regulated kinase signaling
2015, Journal of Biological ChemistryCitation Excerpt :NCX1 works in close partnership with Na,K-ATPase, by utilizing the sodium gradient generated by Na,K-ATPase to drive calcium efflux. Na,K-ATPase has also been shown to function as a motility and tumor suppressor (13, 14) and is involved in the maintenance of epithelial polarity (15) and cell adhesion (16, 17). Moreover, we previously reported that knockdown of Na,K-ATPase β1-subunit (Na,K-β) in MDCK cells led to mesenchymal phenotype accompanied by increased cell proliferation via activation of phosphoinositide-3 kinase (PI3K)/Akt and extracellular-signal-regulated kinase (ERK1/2) pathways (18).
β3 subunit of Na,K ATPase regulates T cell activation with no involvement of Na,K ATPase activity
2015, ImmunobiologyCitation Excerpt :By this method, we were able to detect the β3 subunit in the α subunit depleted lysates, indicating the expression of a free form of the β3 subunit. Self-association of the β subunit has been reported, and this undoubtedly plays a crucial role in cell–cell adhesion (Barwe et al., 2007). It is, therefore, possible that the β3 subunit might form a β3–β3 dimer and carry out other functions beyond its association with the α3 subunit.
- †
S.P.B and S.K. contributed equally to this work.