Cryoelectron microscopy of Na(+),K(+)-ATPase in the two E2P states with and without cardiotonic steroids

Cryoelectron microscopy (cryo-EM) was applied to Na(+),K(+)-ATPase (NKA) to determine the structures of two E2P states, one (E2P(ATP)) formed by ATP and Mg(2+) in the forward reaction, and the other (E2P(Pi)) formed by inorganic phosphate (P(i)) and Mg(2+) in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K(+)-sensitive acceleration of the dephosphorylation reaction is not observed with E2P(Pi), attributed to the presence of a Mg(2+) ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg(2+) in the transmembrane binding cavity is identified only in E2P(Pi), corroborating the idea that it should be denoted as E2P(Pi)·Mg(2+). We can now explain why the absence of transmembrane Mg(2+) in E2P(ATP) confers the K(+) sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2P(ATP) state; CTS binding causes hardly any changes in the structure of NKA, both in E2P(ATP) and E2P(Pi)·Mg(2+), indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the β-subunit and many phospholipid molecules.