Asparagine 706 and glutamate 183 at the catalytic site of sarcoplasmic reticulum Ca2+-ATPase play critical but distinct roles in E2 states.
ABSTRACT Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
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ABSTRACT: Ion translocation by the sarcoplasmic reticulum Ca(2+)-ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 ("A-M3 linker") by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca(2+) and ATP and to form the phosphorylated Ca(2)E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an alpha-helix ("A3 helix") in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca(2)E1P state and thereby to drive the transition to E2P.Journal of Biological Chemistry 04/2009; 284(18):12258-65. · 4.65 Impact Factor
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ABSTRACT: New models of the gastric H,K ATPase in the E1K and E2P states are presented as the first structures of a K+ counter-transport P2-type ATPase exhibiting ion entry and exit paths. Homology modeling was first used to generate a starting conformation from the srCa ATPase E2P form (PDB code 1wpg) that contains bound MgADP. Energy minimization of the model showed a conserved adenosine site but nonconserved polyphosphate contacts compared to the srCa ATPase. Molecular dynamics was then employed to expand the luminal entry sufficiently to allow access of the rigid K+ competitive naphthyridine inhibitor, Byk99, to its binding site within the membrane domain. The new E2P model had increased separation between transmembrane segments M3 through M8, and addition of water in this space showed not only an inhibitor entry path to the luminal vestibule but also a channel leading to the ion binding site. Addition of K+ to the hydrated channel with molecular dynamics modeling of ion movement identified a pathway for K+ from the lumen to the ion binding site to give E2K. A K+ exit path to the cytoplasm operating during the normal catalytic cycle is also proposed on the basis of an E1K homology model derived from the E12Ca2+ form of the srCa ATPase (PDB code 1su4). Autodock analyses of the new E2P model now correctly discriminate between high- and low-affinity K+ competitive inhibitors. Finally, the expanded luminal vestibule of the E2P model explains high-affinity ouabain binding in a mutant of the H,K ATPase [Qiu et al. (2005) J. Biol. Chem. 280, 32349-32355].Biochemistry 05/2007; 46(18):5398-417. · 3.38 Impact Factor
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ABSTRACT: The roles of Ser(72), Glu(90), and Lys(297) at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca(2+)-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca(2+) concentration of phosphorylation by P(i) ("Ca(2+) gradient-dependent E2P formation") showed a reduction of the apparent affinity for luminal Ca(2+) in mutants with alanine or leucine replacement of Glu(90), whereas arginine replacement of Glu(90) or Ser(72) allowed E2P formation from P(i) even at luminal Ca(2+) concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2.BeF(3)(-) complex to luminal Ca(2+). Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu(90) or Ser(72), because the positive charge of the arginine side chain mimics Ca(2+) occupying a luminally exposed low affinity Ca(2+) site of E2P, thus identifying an essential locus (a "leaving site") on the luminal Ca(2+) exit pathway. Mutants with alanine or leucine replacement of Glu(90) further displayed a marked slowing of the Ca(2+) binding transition as well as slowing of the dissociation of Ca(2+) from Ca(2)E1 back toward the cytoplasm, thus demonstrating that Glu(90) is also critical for the function of the cytoplasmically exposed Ca(2+) sites on the opposite side of the membrane relative to where Glu(90) is located.Journal of Biological Chemistry 07/2010; 285(27):20780-92. · 4.65 Impact Factor