| Summary: | The Na +, K+-ATPase is an integral membrane protein that is essential for maintaining the ionic gradient in eukaryotes which is required for many important cellular processes including maintaining cell volume and the secondary transport of solutes. This project investigates the structure, function and physical properties of two classes of inhibitors of the Na+, K+-ATPase, FXYD proteins and the cardiac glycosides. This was achieved with a range of biophysical methods including solid-state NMR. The FXYD proteins are a family of seven physiological inhibitors of the Na+, K+-ATPase named after a shared PFXYD domain. They are transmembrane proteins of 64-178 amino acids which share sequence homology in their transmembrane domains but are sequentiaIIy diverse in their cytoplasmic domains. As the members of the FXYD family are tissue specifically expressed and they affect the activity of the Na +, K+ -ATPase in different ways it is thought that the variable cytoplasmic domains of these proteins are responsible for the unique functional features of each FXYD protein. This work focuses on two FXYD proteins: phospholemman (PLM) which has been linked to cardiac arrhythmia and Mat-S, a marker for several forms of cancer which has two isoforms varying in the lengths of their cytoplasmic domains (short form and long form). The initial hypothesis is that the cytoplasmic region ofPLM interacts with the Na+, K+-ATPase at one or more specific sites which causes a reduction of ATPase activity. This interaction is likely regulated by Ser 68 and/or Ser 63 phosphorylation and occurs in close proximity of the cell membrane surface due to the affinity of the cytoplasmic region of PLM for negatively charged lipid bilayers. This feature is unlikely shared with short form Mat-S as it does not have any identified phosphorylation sites but due to lack of data on long form Mat-S it is not clear whether or not its cytoplasmic region interacts with the Na+, K+-ATPase in a similar way to PLM. Initially, work was undertaken to develop a system to express and purify PLM and Mat-8. An existing method based on the expression of inclusion bodies was first investigated but was found to be inefficient. A new system was therefore developed which successfuIIy enabled the expression and purification of the short form of Mat-S. Further optimisation of this procedure will allow the expression and purification of isotopically labelled Mat-S suitable for analysis by NMR. Synthetic peptides corresponding to the cytoplasmic domains of PLM and Mat-S were studied in isolation of the transmembrane domains in order to investigate their interactions with Na+,K +-ATPase and with phospholipid membrane surfaces. The cytoplasmic domain of PLM has been found to interact with negatively charged phospholipid membrane surfaces and inhibit the Na+, K+-ATPase. These effects are reduced upon phosphorylation of Ser 68 suggesting phosphorylation has a role in regulating inhibition of the Na+, K+-ATPase by PLM. It was found here that the short isoform of Mat-S neither interacts with membrane surfaces nor inhibits Na+,K+-ATPase whereas the long isoform of Mat-S, which has an additional 26 amino acids in the cytoplasmic region, also binds to negatively charged cell membranes and inhibits the Na +, K+ -A'TPase albeit to a smaller extent. Motivated by these results, bioinformatic analysis identified a potential small linear Na", K+-ATPase binding motif; SxxRxS, which is present in PLM, long form Mat-S, several other binding partners of the Na +, K+ -A'TPase but not any other FXYD proteins. This motif in PLM was modeIIed onto a binding site predicted by Anchorsmap, showing both electrostatic and steric compatibility and favouring a site of interaction of the PLM cytoplasmic domain situated close to the membrane surface where interactions with lipid headgroups are possible. The cardiac glycosides are drugs used in heart failure and cardiac arrhythmia which are potent inhibitors of the Na+, K+-ATPase. Solid-state NMR methods were developed to investigate the position of cardiac glycosides in the high-affinity site within native membranes, by exploiting ouabain derivatised in the steroid moiety with a l3C-labeIIed acetonide bridge. By using the solid-state NMR technique of cross- polarisation, the natural abundance l3C nuclei from lipid headgroups and protein side chains can be observed together with a signature peak from the bound ouabain derivative. The paramagnetic broadening agent manganese was titrated into the membranes to probe the depth of the ouabain binding site. Paramagnetic broadening agents shorten the T2 times of local nuclei causing peak broadening with a distance-dependent relationship, thereby having greater effects on more accessible nuclei. The reduction of the ligand peak intensity was compared to the natural abundance l3C signals to estimate the depth of the acetonide bridge. The results suggest that the acetonide group attached to the steroid moiety is close to the surface of the protein. This methodology can now be used with double l3C-labelled ouabain to determine the orientation of the ligand in the binding site. The work presented here provides a range of insights into the structure function relationship of FXYD proteins and reveals a novel method for probing the depths of ligands within transmembrane proteins which can be applied to other systems.
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