Detection of nucleotide binding to Na,K‐ATPase in an aqueous membrane suspension by <sup>13</sup>C cross‐polarization magic‐angle spinning NMR spectroscopy

Middleton, David A.; Jakobsen, Louise Odgaard; Esmann, Mikael

doi:10.1016/j.febslet.2006.11.026

Cited by 4 publications

(11 citation statements)

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“…This is the first time that a kinetic measurement of an active ABC-transporter has been carried out by MAS NMR. To our knowledge, related measurements on a comparably large system with ssNMR have previously only been carried out with 13 C-labelled ATP on Na, K-ATPase ($150 kDa) [32]. In that specific example however, unbound components were filtered out and thus binding rather than kinetics were investigated.…”

Section: Resultsmentioning

confidence: 99%

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

et al. 2008

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The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31 P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150. By analysing time-resolved 31 P spectra, protein activity has been directly assessed. These data demonstrate the general possibility to perform ssNMR studies on a fully active full length ABC transporter and also form the foundation for further kinetic studies on LmrA by NMR.

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Section: Resultsmentioning

confidence: 99%

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

et al. 2008

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“…By contrast, a difference spectrum of [U- 13 Figure 2 A and B). Such differences were seen in an earlier SSNMR study of nucleotide binding to NKA at 4 8C [10] and indicate that the microenvironment or conformation of the nucleotide within the high-affinity site is different to that in the bulk aqueous or membrane phases. The peaks for C4 (149 ppm), C5 (118 ppm) and C6 (156 ppm), which are visible in the direct polarisation spectrum of 4 mm ATP (Figure 2 E), are not detected in the CP-MAS spectra (Figure 2 A-D) because they lack bonded protons that provide the mechanism for efficient cross-polarisation.…”

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confidence: 81%

“…[5][6][7][8] SSNMR studies of bound nucleotide in membranous preparations of NKA from pig kidney and shark salt glands are hampered by a contaminating enzyme activity that rapidly hydrolyses ATP, ADP and AMP in the absence and presence of Mg 2 + . [9,10] Hydrolysis is not inhibited by ouabain, vanadate or adenylate kinase inhibitor. This problem is overcome here by including a Mg 2 + chelator and rapidly freeze-trapping uniformly 13 C/ 15 N-labelled ATP ([U- 13 C, 15 N]ATP) complexed with NKA in membrane preparations.…”

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confidence: 99%

“…[9,10] In so doing we provide insights into the contact surface for ATP within the high-affinity nucleotide site and provide the foundation for further SSNMR studies of bound nucleotide conformation exploiting 13 C-31 P distance measurements, for example.…”

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confidence: 99%

“…Conspicuous peaks include those for C2 and C8 of the adenine ring at 151.4 and 136.7 ppm, respectively, and peaks for the ribose moiety occur in the region from 60.0-90.0 ppm, although the latter overlap the natural abundance signals from the membranes (Figure 1 A). Assignment of the nucleotide peaks to specific positions in the ribose rings was assisted by our previous assignments for bound nucleotide at 4 8C [10] and with a two-dimensional 13 C dipolar assisted rotational resonance (DARR) NMR spectrum (Figure 1 B). The spectrum shows several cross-peaks correlating the ribose ring system, but interactions between the ribose and adenine rings are not detected above the noise.…”

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confidence: 99%

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Solid‐State NMR Studies of Adenosine 5′‐Triphosphate Freeze‐Trapped in the Nucleotide Site of Na,K‐ATPase

et al. 2009

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The Na,K-ATPase (NKA) maintains the cellular potentials of all animal cells by driving the vectorial transport of Na + and K + across the plasma membrane through two different enzyme conformations (E 1 and E 2 ).[1] NKA exhibits high affinity towards the ATP substrate when sufficient Na + is present to induce the E 1 conformation. [2,3] More than fifty years after the discovery of NKA, the details of how ATP interacts with the enzyme in the E 1 conformation remain unknown, and the only X-ray crystal structure of NKA is for the E 2 conformation in the absence of nucleotide.[4] Solid-state NMR spectroscopy (SSNMR) is a valuable alternative to X-ray diffraction for providing atomistic details of ligands bound to receptors within their native membranes.[ C signals from bound ATP and provide preliminary insights into the nucleotide contacts with the high-affinity site.A critical requirement for the SSNMR experiments is to saturate the high-affinity nucleotide site with ATP whilst avoiding excess free or unspecifically bound nucleotide, because SSNMR spectroscopy cannot readily distinguish these from specifically bound nucleotide. Biochemical measurements of radiolabelled nucleotide binding were therefore carried out to establish such a regime. Equilibrium binding curves show nucleotide binding to a single site with dissociation constants in the range of 0.2-0.5 mm, and a capacity of about 2.9 nmol mg À1 protein for both shark and pig NKA in the buffer used for SSNMR experiments ( Figure S1 in the Supporting Information). Samples for SSNMR analysis contain 130 mm NKA, so we can safely assume that the nucleotide site is saturated by adding 160 mm [U-13 C,15 N]ATP to the membranes; this gives a free nucleotide concentration of about 30 mm. The binding assay also demonstrates the absence of additional unspecific binding to NKA. We thus confirm that at these concentrations more than 80 % of the 13 C NMR signal for the nucleotide will correspond to ATP bound specifically to shark or kidney enzyme. Figure 1 A shows 13 C cross-polarisation magic-angle spinning (CP-MAS) SSNMR spectra at À25 8C for a control shark NKA [a] Dr.

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Long-Range Effects of Na⁺ Binding in Na,K-ATPase Reported by ATP

2015

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This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.

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Detection of nucleotide binding to Na,K‐ATPase in an aqueous membrane suspension by ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy

Cited by 4 publications

References 16 publications

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

Solid‐State NMR Studies of Adenosine 5′‐Triphosphate Freeze‐Trapped in the Nucleotide Site of Na,K‐ATPase

Long-Range Effects of Na⁺ Binding in Na,K-ATPase Reported by ATP

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Detection of nucleotide binding to Na,K‐ATPase in an aqueous membrane suspension by 13C cross‐polarization magic‐angle spinning NMR spectroscopy

Cited by 4 publications

References 16 publications

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

Caught in the Act: ATP hydrolysis of an ABC‐multidrug transporter followed by real‐time magic angle spinning NMR

Solid‐State NMR Studies of Adenosine 5′‐Triphosphate Freeze‐Trapped in the Nucleotide Site of Na,K‐ATPase

Long-Range Effects of Na+ Binding in Na,K-ATPase Reported by ATP

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Detection of nucleotide binding to Na,K‐ATPase in an aqueous membrane suspension by ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy

Long-Range Effects of Na⁺ Binding in Na,K-ATPase Reported by ATP