Abstract
We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).
Highlights
Sarcoplasmic reticulum (SR)1 Ca2ϩ-ATPase is a central member of the family of P-type cation-pumping ATPases [1, 2] that actively transport cations by a mechanism involving the formation of a phosphorylated intermediate
We have used alternative methods to identify the cuts as precisely as possible: i) by use of specific antibodies and careful molecular mass estimates based on a number of well known SERCA1a proteolytic fragments from proteinase K or trypsin
Because in this case the migration rate of unknown fragments is compared with that of proteolytic fragments from the same protein, serving as an “ideal standard,” the cleavage points can be rather precisely located; ii) molecular masses were independently measured by several mass spectrometry techniques (MALDI-TOF MS, ESI-Q-TOF, MS/MS)
Summary
Sarcoplasmic reticulum (SR) Ca2ϩ-ATPase is a central member of the family of P-type cation-pumping ATPases [1, 2] that actively transport cations (such as Ca2ϩ, Naϩ, Kϩ, and Hϩ or even heavy metals) by a mechanism involving the formation of a phosphorylated intermediate. It is of interest that similar evidence of a compact E2 conformation accompanying active cation transport has emerged from detailed analysis of the degradation pattern, resulting from Fe2ϩ-catalyzed oxidative cleavages of Naϩ,Kϩ-ATPase (Ref. 17 and references therein and Ref. 18) and Hϩ,Kϩ-ATPase [19] This approach has turned out to give valuable information on the location of Mg2ϩ and ATP1⁄7Mg2ϩ binding sites and suggestive evidence on interactions between cytoplasmic domains during the enzymatic cycle. Since Fe2ϩ can replace Mg2ϩ in its binding site, near the phosphorylation site or in the Mg1⁄7ATP complex, the identification of the various cuts is thought to reflect changes in the position of the Mg2ϩ site(s) in different conformations of the pumping cycle In connection with these studies, the question arises as to the extent that information obtained with one ATPase type such as Naϩ,Kϩ-ATPase, or its close relative Hϩ,Kϩ-ATPase, can be safely applied to other P-type ATPases, including
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