Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state

Abstract

Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

“…The sequence of cytoplasmic Na + binding at the three sites of the Na + ,K + -ATPase is unsettled (4,26). The present data are consistent with the crystal structure-based proposal that the Na + ion at site III is the first ion to bind, followed by occupation of sites I and II, respectively, in a sequential and cooperative binding mechanism (4).…”

“…The relatively minor reduction of the apparent affinity of sites I/II for Na + in C932R could be due to interference with the cooperative interaction between these sites and site III and/or to the tethered cation forcing an order of binding of cytoplasmic Na + different from the normally preferred order. In any case, our data are fully consistent with phosphorylation being controlled by binding of the last Na + to a site different from site III, in agreement with the evolutionary attractive hypothesis proposing that site II, the only transport site conserved in all P-type ATPases, is the last to be occupied from the cytoplasm, triggering the phosphorylation reaction in all P-type ATPases (4,14).…”

“…S1). Crystal structures of the Na + ,K + -ATPase show that two Na + ions bind at sites that overlap substantially with the K + sites (so-called sites I and II), whereas the third Na + ion is bound at a distinct Na + -specific site (site III) (1,4,5). Two isoforms of H + ,K + -ATPase, with different expression profiles, perform electroneutral H + ,K + -exchange by a mechanism basically similar to the Na + ,K + -ATPase mechanism (6,7).…”

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

“…The finding that cholesterol both stabilizes in the presence of SOPS and enhances inhibition by SM or 18:0/18:0 PC suggests that there are more than one binding site. A recent high resolution structure of Na,K-ATPase revealed that cholesterol is bound to at least two sites (17). One cholesterol was observed in the ␣M8 -10 pocket next to the FXYD protein, a location we recently reported to be interacting with SOPS (29) (see also Fig.…”

“…High resolution structures of SERCA, which were obtained for several conformations, revealed that the conformational transitions require substantial movements of TM1-6 3 as well as rearrangements of the three cytoplasmic domains (11). Until now, Na,K-ATPase has been crystallized in only three conformations either from shark rectal gland or pig kidney: E 2 ⅐MgF 4 2Ϫ ⅐2K ϩ (12,13) and E 2 ⅐MgF 4 2Ϫ ⅐2K ϩ ⅐ouabain (14), E 2 P⅐ouabain (15,16), and E 1 ⅐AlF 4 Ϫ ⅐ADP⅐3Na ϩ (17,18).…”