Abstract
Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.
Highlights
Na+/K+-ATPase (EC 3.6.3.9) is an integral membrane protein that transports sodium and potassium ions against an electrochemical gradient
Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of 2 ATP binding sites in the open conformation, the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain
The Na+/K+-ATPase consists of three subunits, the catalytic α-subunit with a molecular mass of about 110kDa, the β-subunit, a glycoprotein with the molecular mass of 40–60kDa and eventually the associated γ-subunit with the molecular mass of 8– 14kDa (Collins & Leszyk 1987; Forbush III et al 1978)
Summary
Na+/K+-ATPase (EC 3.6.3.9) is an integral membrane protein that transports sodium and potassium ions against an electrochemical gradient. It belongs to the family of P-type ATPases that is structurally typified by the L-2-haloacid dehalogenase. Na+/K+-ATPase and Ca2+-ATPase belong to this family and show a high degree of homology, especially at the phosphorylation domain. The tertiary structure of Na+/K+-ATPase has been solved at high resolution by X-ray crystallography(Kanai et al 2013b; Laursen et al 2015; Laursen et al 2013; Morth et al 2011; Morth et al 2007; Nyblom et al 2013a; Ogawa et al 2015; Ogawa et al 2009; Shinoda et al 2009; Yatime et al 2011) and partially several N-domain structures by X-ray crystallography (Håkansson 2003) and NMR(Mark Hilge 2003). The ion transport of Na+ and K+ catalyzed by Na+/K+-ATPase in this subunit is believed to occur via transition between two major conformational states, the
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