Active conformations of neuronal Na+, K+-ATPase isoforms and a disease-causing mutant

Figures

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  Fig. 1. Structures of Na+ -bound states of α3-Na+,K+-ATPase. A Sodium-bound cryo-EM structure of wt α3ß1FXYD1 Na+,K+-ATPase with α-A domain in yellow, α-P domain in blue, α-N domain in red, ß-subunit in orange, FXYD1 in light green(barely visible in the back), and the α-TM-domain in gray, teal and purple, respectively, for the three indicated states. B cryo-EM density around the Na+ binding sites I, II and III of the respective states shown in A (contoured at 4 s.d. for Na2E1-ATP and [Na3]E1P-ADP and at 3.5 s.d. for [Na3]E2P). Site III only shows density for a bound ion in the occluded [Na3]E1PADP and [Na3]E2P states. Hence, we propose a binding mechanism occupying first exposed binding sites I and II, then with internal reorganization leading to occupation of site III, cooperative binding of a third Na+ at exposed sites I/II and occlusion/phosphorylation. In [Na3]E2P, site III is shifted towards M8 with D923. We propose that the shift breaks the cooperativity of the three sites and initiates extracellular release. C Cryo-EM density around the phosphorylation and nucleotide binding sites (contoured at 5.5 s.d. for Na2E1-ATP, 7 s.d. for [Na3]E1P-ADP, and at 6 s.d. for [Na3]E2P). D…

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  Fig. 2. E2P states of α1 and α3 Na+,K+-ATPase. A Structure and cryoEM map of wt α3ß1FXYD1-Na+,K+-ATPase. Colors as in Fig. 1, except the α-TM domain is now in green. B Cryo-EM density around the phosphorylation site contoured at 7 s.d. C Major rearrangements of the [Na3]E2P to E2P transition showing the rearrangement of TM1-5 and alignment of the cytoplasmic domains, which rotate in a coordinated 15° angle. D Density of the cytoplasmic domains of wt α1 and α3 isoforms in the [Na3]E2P versus outward open E2P states at a contour level of 2.5. The density likely representing the far N-terminus of the A-domain, which extends towards the saposin A nanodisc, is highlighted in orange. Other colors as in (A). This density is only visible in α1 but not in α3, while the N-domain is better defined in α3 versus α1. E E1/E2 equilibria of FITC-labeled Na, K-ATPase isoforms estimated from the amplitude of fluorescence changes upon addition of 20 mM RbCl and then 100 mM NaCl. The data represent averages of four biological determinations ± SEM. Values of E1/(E1 + E2) in the Na- and K-free high ionic strength medium are as follows: α1β1 0.86 ± 0.1; α2β1 0.67 ± 0.02; α3β1 0.33 ± 0.03 (p = 3e-4 for α1β1 vs…

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  Fig. 3. Characterization of the Q140L AHC variant. A Alignment of wt α3 and α3-Q140L showing overall similar structures for all four obtained states, except a slight rotation of the cytoplasmic domains in [Na3]E2P. B Lipid density for wt and Q140L at a contour level of 6 (orange) and 4 (gray). C specific activity of wt and mutant Na+,K+-ATPases purified in the presence of different lipid mixtures. The average of 5 biological replicates ±SEM is shown with p-values determined from a one-sided t-test. D Inflection temperatures of wt and Q150L α1-Na, K-ATPase (equivalent of α3-Q140L) in the conformations shown on the x-axis. Additives in brackets were used to stabilize the conformation, as shown in suppl. Fig. 1. Average inflection temperatures of 3-6 biological replicates ±SEM are shown with p-values determined from a two-sided t-test for respective conditions.

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  Fig. 4. FITC-labeled Na+,K+-ATPase (α1β1). Cation titrations of fluorescence changes. The data represent averages ± SEM of three determinations at each Rb or Na concentration. MgCl2 was omitted from the medium in this experiment. Raised K1/2Rb with lower K1/2Na for Q150L versus WT, indicates relative poise of the E1/E2 conformational equilibrium towards E1. For α1WT, for PS + PE versus PS, a similar conclusion pertains, but for the Q150L, the similar K1/2Rb values for PS + PE versus PS suggest most simply that binding of PE is impaired. K1/2 values representing averages of three biological replicates ±SEM are as follows: A, B wt K0.5Rb 0.43 ± 0.02; Q150L K0.5Rb 2.78 ± 0.27 p = 0.001 (two-sided t-test); wt K0.5Na 16.0.0 ± 1.0; Q150L K0.5 Na 6.6 ± 2.3, p = 0.02. C, D wt PS K0.5 Rb 0.43 ± 0.02; PS + PE K0.5 Rb 1.12 ± 0.048, p = 2e-4; Q150L K0.5 Rb 2.78 ± 0.27; PS + PE K0.5 Rb 3.73 ± 0.29, p = NS).

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  Fig. 5. Schematic representation of principal rearrangements between structures obtained in the presence of Na+ and ATP. Arrows indicate changes to the previous conformation. Coloring as in previous figures. The three principal transitions are (1) binding of the 3rd Na-ion pushes the two bound Na-ions to their neighboring sites, causing closure of the cytoplasmic gate and ion occlusion and causes the cytoplasmic domains to form a tighter arrangement, allowing phosphorylation from ATP, (2) The E1P- > E2P conformational transition causes an immediate rearrangement of the A and N-domain, with the N-domain rotating backward, allowing the release of ATP and the A-domain rotation to cover the aspartyl-phosphate with its signature TGES- loop. The transmembrane domain remains largely unaltered except for destabilization of site III and the straightening of TM1. (3) Destabilization of site III and release of strain causes the rearrangement of TM1-6 while A, N and P-domains rotate in a concerted action.