Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

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Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42652-4 ◽

1984 ◽

Vol 259 (23) ◽

pp. 14651-14656 ◽

Cited By ~ 10

Author(s):

S Highsmith ◽

A J Murphy

Keyword(s):

Sarcoplasmic Reticulum ◽

Binding Site ◽

Binding Sites ◽

Calcium Binding ◽

Atp Binding ◽

Atp Binding Site ◽

High Affinity ◽

Calcium Binding Sites

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Identification and mechanistic analysis of an inhibitor of the CorC Mg2+ transporter

10.1101/2021.02.10.430528 ◽

2021 ◽

Author(s):

Yichen Huang ◽

Kaijie Mu ◽

Xinyu Teng ◽

Yimeng Zhao ◽

Yosuke Funato ◽

...

Keyword(s):

Binding Site ◽

Transmembrane Domain ◽

Chemical Compounds ◽

Cytoplasmic Domain ◽

Structural Basis ◽

Atp Binding ◽

Inhibitory Effects ◽

Atp Binding Site ◽

Design And Optimization ◽

Mechanistic Analysis

AbstractThe CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial member of the CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. While CorC and CNNM have attracted interest as therapeutic targets, inhibitors targeting the ir regulatory ATP-binding site have not yet been identified.Here, we performed a virtual screening of CorC by targeting its regulatory ATP-binding site, identified a chemical compound named IGN95a with inhibitory effects on both ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more towards the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift. Our results provide a structural basis for the further design and optimization of chemical compounds targeting the regulatory ATP-binding site of CorC as well as mechanistic insights into how ATP and chemical compounds modulate the transport activity of CorC.

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Labeling of lysine 492 with pyridoxal 5'-phosphate in the sarcoplasmic reticulum Ca(2+)-ATPase. Lysine 492 residue is located outside the fluorescein 5-isothiocyanate-binding region in or near the ATP binding site.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)36876-0 ◽

1993 ◽

Vol 268 (28) ◽

pp. 20930-20936

Author(s):

K Yamagata ◽

T Daiho ◽

T Kanazawa

Keyword(s):

Sarcoplasmic Reticulum ◽

Binding Site ◽

Atp Binding ◽

Binding Region ◽

Atp Binding Site

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The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP

Biochemical Journal ◽

10.1042/bj20030740 ◽

2004 ◽

Vol 377 (1) ◽

pp. 95-105 ◽

Cited By ~ 13

Author(s):

Juha OKKERI ◽

Liisa LAAKKONEN ◽

Tuomas HALTIA

Keyword(s):

Escherichia Coli ◽

Sarcoplasmic Reticulum ◽

Binding Site ◽

Molecular Model ◽

Nucleotide Binding ◽

Atp Binding ◽

Atp Binding Site ◽

Disease Mutations ◽

Disease Protein ◽

P Type

In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain. The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx. 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase. None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA. However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases. This motif is also found in Wilson disease protein where several disease mutations cluster in it. In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503→Ser), G505R and A508F of ZntA. At low [ATP], these mutant ATPases are poorly phosphorylated. The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher [ATP], suggesting that these mutations lower the affinity for ATP. In all three mutant ATPases, phosphorylation by Pi has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding. In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template. In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site. In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation.

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