Transport mechanism and membrane interactions of the cation pumps

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• Poul Nissen, Aarhus University, Denmark
• Thursday 17 February 2011, 16:15-18:00
• Max Perutz Lecture Theatre, Medical Research Council (MRC) (MRC Laboratory of Molecular Biol.

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Danish National Research Foundation, Center for Membrane Pumps in Cells and Disease – PUMPKIN . Department of Molecular Biology, Aarhus University, Denmark, DK – 8000 Aarhus C P-type ATPases encompass the cation pumps like Na+,K+-ATPase and Ca2+-ATPase. They couple the transport and countertransport of ions, through a membrane spanning domain, to ATP hydrolysis via formation and breakdown of a phosphoenzyme intermediate at cytoplasmic domains. The enzymes undergo large conformational changes where half-channels open and close, and where the ion binding sites at the middle of the membrane switch their specificity and orientation (Møller 2010). Worth noting, these large-scale movements take place in close interaction with the membrane. We study P-type ATPases by interdisciplinary approaches ranging from crystal structures to model organisms. Na+,K+-ATPase is of key importance in animal cells and physiology. The enzyme transports three sodium ions out and two potassium in per ATPase cycle. From the crystal structure of the pig kidney enzyme in the potassium-bound form – further probed by mutagenesis and fast-kinetics – we pinpointed a key role of the alpha subunit C-terminus, and specifically on sodium binding (Morth et al. 2007). The underlying mechanisms however remained elusive. Furthermore, the uneven and electrogenic transport stoichiometry leads to fundamental questions of how an unoccupied site is accounted when releasing sodium and switching to potassium countertransport. Using electrophysiology, MD simulations and structural analysis we have reached a new model of Na+,K+-ATPase function: A C -terminal ion pathway, plugged by the C-terminus, leads to the ion binding sites at the conserved Asp926 residue (pig alpha1 isoform, Asp930 in human alpha2 isoform) which in the potassium-bound state becomes protonated (Poulsen et al. 2010). Through this channel the proton can again return to the cytoplasm upon sodium binding, thus ensuring that the overall 3:2 transport mechanism becomes electrogenic. The importance is further underscored by a large cluster of mutations at the C-terminal region associated with neurological diseases, e.g. familial hemiplegic migraine 2 (FHM2) and rapid-onset dystonia with parkinsonism (RDP). A low resolution crystal structure of the phosphorylated E2P form of the Na+,K+-ATPase in complex with the cardiotonic steroid ouabain shows conformational changes that not only explain the mechanism of inhibition, but also hints at the possible role of these steroids in signaling with Na+,K+-ATPase working as a receptor (Yatime et al. 2010). In favourable crystal forms of the related Ca2+-ATPase we have observed electron density for lipid-detergent bilayers between molecules and we compare this experimental basis to molecular dynamics simulations and biochemical/biophysical data on the protein-lipid interaction thus to address a working transport process across a membrane (Sonntag et al. submitted).¨ Cu(I)-ATPases constitute another important P-type ATPase subfamily. We have determined the first crystal structure of a Cu(I)-ATPase in a copper released state providing critical new insight on the copper transport pathway (Gourdon et al. submitted).

References: Morth JP, Pedersen BP, Toustrup-Jensen M, Andersen JP, Vilsen B, Nissen P (2007). Crystal structure of the sodium-potassium pump. Nature 450, 1043-1049 Møller JV, Olesen C, Winther AM, Nissen P (2010). The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump. Q Rev Biophys. 2010 Sep 1:1-66 (epub ahead of print) Poulsen H, Khandelia H, Morth JP, Bublitz M, Mouritsen OG, Egebjerg J, Nissen P (2010). Neurological disease mutations compromise a C-terminal ion pathway in the Na()/K()-ATPase. Nature 467, 99-102 Yatime L, Laursen M, Morth JP, Esmann M, Nissen P, Fedosova NU (2010). Structural insights into the high affinity binding of cardiotonic steroids to the Na(),K()-ATPase. J. Struct. Biol. 2010 Dec. 20 (epub ahead of print)

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