The complex ATP–Fe ²⁺ serves as a specific affinity cleavage reagent in ATP-Mg ²⁺ sites of Na,K-ATPase: Altered ligation of Fe ²⁺ (Mg ²⁺ ) ions accompanies the E ₁ P→E ₂ P conformational change

Abstract

In the presence of ascorbate͞H2O2, ATP-Fe 2؉ or AMP-PNP-Fe 2؉ complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP-Mg 2؉ sites. The cleavages reveal contact points of Fe 2؉ or Mg 2؉ ions. In E1 and E 1Na conformations, two major cleavages are detected within the conserved 708 TGDGVNDSPALKK sequence (at V712 and nearby), and one (E1Na) or two (E1) minor cleavages near V440. In media containing sodium and ATP, Fe 2؉ A long series of experiments established the Post-Albers kinetic mechanism of Na,K-ATPase and related P-type cation pumps (1-3). Active Na ϩ and K ϩ transport involves (i) Na cyt -dependent phosphorylation from ATP, and Na ϩ occlusion, E 1 3E 1 P(Na); (ii) Na ϩ transport outward across the membrane coupled to E 1 P3E 2 P; (iii) K exc -activated dephosphorylation, and occlusion, E 2 P3E 2 (K); and (iv) K ϩ transport inward across the membrane coupled to E 2 (K)3E 1 , accelerated by ATP acting with low affinity. For other pumps, steps i or iii are activated by the appropriate cations, which are transported in steps ii or iv.Despite our extensive knowledge of function, a proper understanding of active transport cannot be achieved without knowledge of molecular structure. In this regard, the recent publication of the 2.6-Å crystal structure of sarcoplasmic reticulum Ca-ATPase is an event of unparalleled importance (4). The structure confirms the existence of ten transmembrane helices deduced for Ca, Na,K Ϫ , H,K Ϫ , and H-pumps by biochemical techniques (5) and reveals several unexpected features, including distortion of the M4 and M6 membrane-spanning helices involved in occluding Ca ions. The details of Ca occlusion sites fit well with those deduced in extensive mutagenesis studies (3, 6). The cytoplasmic sector of the pump is divided into three domains, two domains N (nucleotide) and P (phosphorylation) within the loop between M4 and M5, well separated from a third A (actuator or anchor) domain containing the loop between M2 and M3 and the segment leading into M1. The fold of the P domain is like that of haloacid dehydogenase and related proteins with homologies to P-type pumps in conserved cytoplasmic sequences (7,8). Comparison of the crystal structure (an E 1 ⅐Ca conformation) with cryoelectron microscope images of Ca-ATPase in both E 1 or E 2 conformations (9), suggested that, in the change from E 1 to E 2 , domain A makes contact with the P͞N domain (see below). Presumably, the tertiary structures of other P-type pumps will resemble that of Ca-ATPase, particularly within the cytoplasmic domains, but will show detailed differences related to the cation specificities, and for Na,K-ATPase and H,K-ATPase to the presence of a ␤ subunit .Important as it is, the crystal structure of a pump in one conformation cannot provide full structural information on other conformations. Recently, we described a technique of specific oxidative cleavage of renal Na,K-ATPase, which utilizes Fe 2ϩ ͞ascorbate͞H 2 O 2 and provides unique inform...

Selective Fe2+-catalyzed Oxidative Cleavage of Gastric H+,K+-ATPase

“…This indicates that an excess of uncomplexed ATP is able to compete with and displace the ATPFe 2ϩ complex from the site and so prevent the cleavages (see Ref. 28). In addition, the presence of an excess of Mg 2ϩ ions also suppressed the fragments at VNDS and near VIGDA, as would …”

“…Because more than one peptide bond can be cleaved from the same metal site, the technique has the important feature of providing information on proximity of cleaved segments in the native protein. In the case of cleavage by the ATP-Fe 2ϩ complex, the properties indicate that the Fe 2ϩ ion is ligated by residues normally occupied by Mg 2ϩ ions, and thus cleavages reveal contacts of Fe 2ϩ or Mg 2ϩ ions (28). Cleavages catalyzed by Fe 2ϩ ions and the ATP-Fe 2ϩ complex have provided evidence that both E 1 3 E 2 and E 1 P 3 E 2 P conformational changes are associated with large movements in the cytoplasmic domains and also with a change in ligation of Mg 2ϩ ions between E 1 P and E 2 P (26 -28).…”

Distinct Natures of Beryllium Fluoride-bound, Aluminum Fluoride-bound, and Magnesium Fluoride-bound Stable Analogues of an ADP-insensitive Phosphoenzyme Intermediate of Sarcoplasmic Reticulum Ca2+-ATPase

“…Furthermore in the Ca 2ϩ -ATPase, the A domain, which is not present in the phosphoserine phosphatase, is also involved in the formation of the catalytic site in E2P by its gathering with the P and N domains upon the E1P to E2P transition (10 -14, 62). The TGES 184 outermost loop on the A domain was, in fact, predicted to be essentially involved (4,(62)(63)(64). It is likely that the interactions between the three cytoplasmic domains are somewhat rearranged (yet in a compactly organized state) upon the change from the tetrahedral ground state to the trigonal bipyramidal transition state and, thus, upon the associated possible rearrangements in coordination of the bound phosphate during the E2P hydrolysis and, further, it is likely that such changes rearrange the connected transmembrane helices so as to close the postulated Ca 2ϩ release pathway and gate.…”

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

“…A, the amounts of EP formed with 32 The extensive hydrogen bond network is actually formed between the TGES 184 loop, the Asp 601 -Pro 603 loop, Asp 627 , the residues in Lys 352 -Asn 359 , and Arg 560 on N domain, and stabilizing the associated state of the domains. Asp 703 and Glu 183 on the TGES 184 loop were predicted by the iron-catalyzed cleavage of Na ϩ /K ϩ -ATPase (38) to participate together in the Mg 2ϩ binding in E2P. These facts, together with the observed blocking of the E1P to E2P transition by the specific substitutions, strongly suggest that the above residues (Asp 601 , Pro 603 , Asp 627 , and Asp 703 ) or at least some of them are involved in the A-P domain interaction together with the TGES 184 loop for the loss of the ADP sensitivity, although information on the threedimensional structure of E2PCa 2 formed in step 4 (the ADPinsensitive EP with bound or occluded (39) Ca 2ϩ ) is yet unavailable.…”

“…the ATP binding and phosphorylation, the isomeric transition of EP (the domain association), and its hydrolysis. Lys 684 seems not to be involved in the domain interaction in E2P (E2V), but likely functions in coordination of the covalently bound phosphate in EP (38,40) and thus in the possible change in the immediate vicinity of the phosphate for the loss of ADP sensitivity.…”