P-type pumps are the most flexible molecular machines know with every part changing position during the catalytic cycle. How a single polypeptide can carry out such remarkable motion and pump ions against a steep membrane potential and concentration gradient have remained puzzling problems. In the first 5-year period (April 2007 - March 2012), PUMPkin elucidated some of the most difficult-to-solve structures of these pumps, namely basic aspects of structure and function of the sodium-potassium pump, the plasma membrane proton pump, and the copper pumps, besides a completion of the calcium pump cycle. With 3D structures of the most important pumps in key conformations, the basic mechanisms is now approached in detail and its application to a variety of transport substrates and inhibitors is emerging. This is a remarkable achievement in that new P-type pump structure have so far been recalcitrant to structural studies by other laboratories except that of Chikashi Toyoshima, who solved the calcium pump structure in 2000. Functional and mutational studies have supported the discoveries found and we have initiated studies on new pumps. In the first period, five Nature papers and a wealth of other articles in highly reputed journals (exceeding 1000 in 2010) as well as two awarded patents and the spin-out company Pcovery mark the results of the PUMPkin team.
PUMPkin has been an outstanding success and with our second period (April 2012 - March 2017) we will aim for even higher levels of cutting-edge research for the benefit of Danish society and new science.
Many details of the basic pump mechanism are still not elucidated and to complete the picture, direct visualization and analysis of single pump molecules in action will be approached. We also wish to proceed along new avenues of integral studies of cellular assemblies and higher order hierarchical structures of membranes. At this new level, we wish to understand pump function in the context of the cellular network created by multiple other proteins, lipids, metabolites and nucleic acids. How these interactions regulate pump activity and are translated into a physiological phenotype and response will be studied by a broad palette of methods in cell biology and whole organism biology, yet with the focus an coherence offered by P-type ATPase at center stage. We also wish to determined higher order structures of well-characterized pumps in assembly with other cellular components by pushing further emerging techniques such as small-angle x-ray scattering, single-particle EM and EM tomography.
Another important path for PUMPkin in the next 5-year period will be to study the function and mechanism of "orphan pumps." The P4- and P5-ATPase clades have 19 members in the human genome, comparable to 15 well-known ion pumps. The presence of multiple isoforms underscores the important function of P4- and P5-ATPases, but we hardly know what they do as even expression and purification is difficult. Through a focused effort we will combine established methods with new biophysical tools such as giant liposomes (GUV) and nanodisc reconstitution in order to crack the code that prevented these pumps from being studied and properly accounted for in human cell biology and physiology. Also orphan P-type ATPase transporters from bacteria and fungi will be exploited.
Besides these lines of novel and highly promising research we wish to continue with in-depth investigation of the pumps we already have characterized in some detail. This involved research in the structure, function, cell biology and regulation of ion pumps employing many of the same methods we already have established at PUMPkin. This ensures optimal utilization of the resources available and provides important training opportunities for doctoral students as well as important contact point to laboratories world wide.
Photos from the annual PUMPkin summer meeting 2012