Importance of conserved α-subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase

The segment (708)TGDGVNDSPALKK(720) in the cu subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg2+ or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713) ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp710 contributes to coordination of Mg2+ during transfer of gamma -phosphate to Asp(369) in the high energy (MgE1P)-E-.[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg2+ binding in the E(2)P(.)ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). Th, Asp(710), Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from Pi of the E(2)(.)ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710), Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of T1(+) 2- to 3-fold, suggesting a role in transmission of KC stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.