Abstract
In human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. Yet, the overall shape of P5B-ATPases and the mechanisms of polyamine recognition, uptake and transport remain elusive. Here we describe a series of cryo-electron microscopy structures of a yeast homolog of human ATP13A2-5, Ypk9, determined at resolutions reaching 3.4 Å, and depicting three separate transport cycle intermediates, including spermine-bound conformations. Surprisingly, in the absence of cargo, Ypk9 rests in a phosphorylated conformation auto-inhibited by the N-terminus. Spermine uptake is accomplished through an electronegative cleft lined by transmembrane segments 2, 4 and 6. Despite the dramatically different nature of the transported cargo, these findings pinpoint shared principles of transport and regulation among the evolutionary related P4-, P5A- and P5B-ATPases. The data also provide a framework for analysis of associated maladies, such as Parkinson’s disease.
Highlights
In human cells, P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders
A cryo-electron microscopy (cryo-EM) structure, coined E2Pinhib, was determined at an average resolution of 3.5 Å, generated in the presence of the phosphate analog beryllium fluoride (BeF3−), which has been previously employed as a conformationstabilizing tool for investigations of P-type ATPases[17] (Methods and Supplementary Table 1)
The cryo-EM maps show wellresolved domains (Supplementary Figs. 1a and 2), enabling de novo model building of the entire ATPase, except for certain peripheral loops of the soluble domains, and some 200 Nterminal residues that are largely absent in the human P5BATPase members (Supplementary Fig. 3)
Summary
P5B-ATPases execute the active export of physiologically important polyamines such as spermine from lysosomes to the cytosol, a function linked to a palette of disorders. The structure reveals a typical P-type ATPase fold, including the conserved cytosolic actuator (A-), phosphorylation (P-), and nucleotide-binding (N-) domains, and a transmembrane (M-) domain composed of ten transmembrane spanning helices, TM1–10 (Fig. 1). An N-terminal domain (NTD) structurally related to the one observed for the yeast P5A-ATPase Spf[1] is detected[4] (Supplementary Fig. 4).
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