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bioRxiv · 10.1101/2024.08.13.607722

In silico studies provide new structural insights into trans-dimerization of β1 and β2 subunits of the Na+,K+-ATPase

Ramirez Salinas, G.·Rosas-Trigueros, J. L.·Sosa Huerta, C.·Shoshani, L.·Martinez Archundia, M. T.
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Abstract

The Na+,K+-ATPase is an electrogenic transmembrane pump located in the plasma membrane of all animal cells. It is a dimeric protein composed of and {beta} subunits and has a third regulatory subunit ({gamma}) belonging to the FXYD family . This pump plays a key role in maintaining low concentration of sodium and high concentration of potassium intracellularly. The subunit is the catalytic one while the {beta} subunit is important for the occlusion of the K+ ions and plays an essential role in trafficking of the functional {beta} complex of Na+,K+-ATPase to the plasma membrane. Interestingly, the {beta}1 and {beta}2 (AMOG) isoforms of the {beta} subunit, function as cell adhesion molecules in epithelial cells and astrocytes, respectively. Early experiments suggested a heterotypic adhesion for the {beta}2. Recently, we reported a homotypic trans-interaction between {beta}2-subunits expressed in CHO cells. In this work we use In Silico methods to analyze the physicochemical properties of the putative homophilic trans-dimer of {beta}2 subunits and provide insights about the trans-dimerization interface stability. Our structural analysis predicts a molecular recognition mechanism of a trans-dimeric {beta}2-{beta}2 subunit and permits designing experiments that will shed light upon possible homophilic interactions of {beta}2 subunits in the nervous system. Author summaryThe adhesion molecule on glia (AMOG) is the {beta}2 isoform of the {beta}-subunit of the Na+-pump that is localized in the nervous system, specifically in astrocytes. It was shown that it mediates Neuron-Astrocyte interaction, promoting neurite outgrowth and migration during brain development. In recent years we have shown that the ubiquitous {beta}1 isoform is a homophilic adhesion molecule in epithelia and therefore we hypothesized that {beta}2 could also interact as a homophilic adhesion protein. In a previous work we show that fibroblasts (CHO) transfected with the human {beta}2 subunit of the Na+-pump become adhesive. Moreover, protein-protein interaction assay in a co-culture of cells transfected with {beta}2 tagged with two different markers (His6 and YFP) reveal a positive interaction between the {beta}2-subunits. In the present work, we apply bioinformatics methods to analyze and discuss the formation of a trans-dimer of {beta}2-subunits. Our In Silico study predicts a relatively stable dimer with an interface that involves the participation of four out of the seven N-glycosylation sites. Nevertheless, interacting interface and the dynamics of the {beta}2-{beta}2 trans-dimer is different from that of the {beta}1-{beta}1 dimer; it involves different surfaces and therefore it explains why {beta}-subunits can not form mixed ({beta}1-{beta}2) trans-dimers.

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Ramirez Salinas, G., Rosas-Trigueros, J. L., Sosa Huerta, C., Shoshani, L., Martinez Archundia, M. T.. 2024-08-16. In silico studies provide new structural insights into trans-dimerization of β1 and β2 subunits of the Na+,K+-ATPase. https://doi.org/10.1101/2024.08.13.607722

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