Journal of Molecular Biology
Volume 365, Issue 3, 19 January 2007, Pages 706-714
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Janus Model of The Na,K-ATPase β-Subunit Transmembrane Domain: Distinct Faces Mediate α/β Assembly and β-β Homo-oligomerization

https://doi.org/10.1016/j.jmb.2006.10.029Get rights and content

Abstract

Na,K-ATPase is a hetero-oligomer of α and β-subunits. The Na,K-ATPase β-subunit (Na,K-β) is involved in both the regulation of ion transport activity, and in cell–cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-β transmembrane domain (TMD) that could mediate protein–protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-β with Na,K-α, and the glycine zipper face is involved in the homo-oligomerization of Na,K-β. Point mutations in the heptad repeat motif reduced Na,K-β binding to Na,K-α, and Na,K-ATPase activity. Na,K-β TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell–cell adhesion. These results provide a structural basis for understanding how Na,K-β links ion transport and cell-cell adhesion.

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.

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    S.P.B and S.K. contributed equally to this work.

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