Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405, 647–655 (2000)
Sørensen, T. L., Møller, J. V. & Nissen, P. Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304, 1672–1675 (2004)
Toyoshima, C., Nomura, H. & Tsuda, T. Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature 432, 361–368 (2004)
Jensen, A. M., Sørensen, T. L., Olesen, C., Møller, J. V. & Nissen, P. Modulatory and catalytic modes of ATP binding by the calcium pump. EMBO J. 25, 2305–2314 (2006)
Olesen, C. et al. The structural basis of calcium transport by the calcium pump. Nature 450, 1036–1042 (2007)
Winther, A. M. et al. The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm. Nature 495, 265–269 (2013)
Andersson, M. et al. Copper-transporting P-type ATPases use a unique ion-release pathway. Nat. Struct. Mol. Biol. 21, 43–48 (2014)
Wang, K. et al. Structure and mechanism of Zn2+-transporting P-type ATPases. Nature 514, 518–522 (2014)
Møller, J. V., Olesen, C., Winther, A. M. & Nissen, P. The sarcoplasmic Ca2+-ATPase: design of a perfect chemi-osmotic pump. Q. Rev. Biophys. 43, 501–566 (2010)
Sørensen, T. L., Dupont, Y., Vilsen, B. & Andersen, J. P. Fast kinetic analysis of conformational changes in mutants of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 275, 5400–5408 (2000)
Wakabayashi, S. & Shigekawa, M. Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase. J. Biol. Chem. 259, 4427–4436 (1984)
Mahaney, J. E., Thomas, D. D. & Froehlich, J. P. The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model. Biochemistry 43, 4400–4416 (2004)
Forbush, B. III . Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP. J. Biol. Chem. 262, 11104–11115 (1987)
Holmgren, M. et al. Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase. Nature 403, 898–901 (2000)
Raimunda, D., González-Guerrero, M., Leeber, B. W., III & Argüello, J. M. The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function. Biometals 24, 467–475 (2011)
Zhao, Y. et al. Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue. Nature 474, 109–113 (2011)
Juette, M. F. et al. Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. Nat. Methods 13, 341–344 (2016)
Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008)
Zhao, Y. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010)
Faxén, K. et al. Characterization of a Listeria monocytogenes Ca2+ pump: a SERCA-type ATPase with only one Ca2+-binding site. J. Biol. Chem. 286, 1609–1617 (2011)
Andersen, J. L., Gourdon, P., Møller, J. V., Morth, J. P. & Nissen, P. Crystallization and preliminary structural analysis of the Listeria monocytogenes Ca2+-ATPase LMCA1. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 67, 718–722 (2011)
Dyla, M. et al. Engineering a prototypic P-type ATPase Listeria monocytogenes Ca2+-ATPase 1 for single-molecule FRET studies. Bioconj. Chem. 27, 2176–2187 (2016)
Kotšubei, A., Gorgel, M., Morth, J. P., Nissen, P. & Andersen, J. L. Probing determinants of cyclopiazonic acid sensitivity of bacterial Ca2+-ATPases. FEBS J. 280, 5441–5449 (2013)
Rosch, J. W., Sublett, J., Gao, G., Wang, Y. D. & Tuomanen, E. I. Calcium efflux is essential for bacterial survival in the eukaryotic host. Mol. Microbiol. 70, 435–444 (2008)
Juette, M. F. et al. The bright future of single-molecule fluorescence imaging. Curr. Opin. Chem. Biol. 20, 103–111 (2014)
Zheng, Q. et al. Ultra-stable organic fluorophores for single-molecule research. Chem. Soc. Rev. 43, 1044–1056 (2014)
Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β2AR–G-protein activation. Nature 547, 68–73 (2017)
Vafabakhsh, R., Levitz, J. & Isacoff, E. Y. Conformational dynamics of a class C G-protein-coupled receptor. Nature 524, 497–501 (2015)
Wakabayashi, S., Ogurusu, T. & Shigekawa, M. Participation of H+ in the Ca2+-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase. Biochemistry 29, 10613–10620 (1990)
Musgaard, M., Thøgersen, L. & Schiøtt, B. Protonation states of important acidic residues in the central Ca2+ ion binding sites of the Ca2+-ATPase: a molecular modeling study. Biochemistry 50, 11109–11120 (2011)
Lund, S. & Møller, J. V. Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer. J. Biol. Chem. 263, 1654–1664 (1988)
Marchand, A. et al. Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca2+ -ATPase reveals key events in phosphorylation and Ca2+ release. J. Biol. Chem. 283, 14867–14882 (2008)
Anthonisen, A. N., Clausen, J. D. & Andersen, J. P. Mutational analysis of the conserved TGES loop of sarcoplasmic reticulum Ca2+-ATPase. J. Biol. Chem. 281, 31572–31582 (2006)
Møller, J. V., Lind, K. E. & Andersen, J. P. Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein–protein interactions. J. Biol. Chem. 255, 1912–1920 (1980)
Makinose, M. & Hasselbach, W. ATP synthesis by the reverse of the sarcoplasmic calcium pump. FEBS Lett. 12, 271–272 (1971)
Daiho, T., Yamasaki, K., Danko, S. & Suzuki, H. Critical role of Glu40–Ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme. J. Biol. Chem. 282, 34429–34447 (2007)
Pilotelle-Bunner, A., Cornelius, F., Sebban, P., Kuchel, P. W. & Clarke, R. J. Mechanism of Mg2+ binding in the Na+,K+-ATPase. Biophys. J. 96, 3753–3761 (2009)
Picard, M. et al. Ca2+ versus Mg2+ coordination at the nucleotide-binding site of the sarcoplasmic reticulum Ca2+-ATPase. J. Mol. Biol. 368, 1–7 (2007)
Albers, R. W. Biochemical aspects of active transport. Annu. Rev. Biochem. 36, 727–756 (1967)
Post, R. L., Hegyvary, C. & Kume, S. Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase. J. Biol. Chem. 247, 6530–6540 (1972)
Dode, L. et al. Functional comparison between secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and sarcoplasmic reticulum Ca2+-ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses. J. Biol. Chem. 280, 39124–39134 (2005)
Veshaguri, S. et al. Direct observation of proton pumping by a eukaryotic P-type ATPase. Science 351, 1469–1473 (2016)
Mathiasen, S. et al. Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes. Nat. Methods 11, 931–934 (2014)
Baginski, E. S., Foà, P. P. & Zak, B. Microdetermination of inorganic phosphate, phospholipids, and total phosphate in biologic materials. Clin. Chem. 13, 326–332 (1967)
Weber, K. & Osborn, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412 (1969)
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)
Blanchard, S. C., Kim, H. D., Gonzalez, R. L., Jr, Puglisi, J. D. & Chu, S. tRNA dynamics on the ribosome during translation. Proc. Natl Acad. Sci. USA 101, 12893–12898 (2004)
Lata, S., Reichel, A., Brock, R., Tampé, R. & Piehler, J. High-affinity adaptors for switchable recognition of histidine-tagged proteins. J. Am. Chem. Soc. 127, 10205–10215 (2005)
Akyuz, N. et al. Transport domain unlocking sets the uptake rate of an aspartate transporter. Nature 518, 68–73 (2015)
Qin, F. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling. Biophys. J. 86, 1488–1501 (2004)
We thank J. V. Møller and the members of the Blanchard and Nissen laboratories for helpful discussions, A. M. Nielsen, T. Klymchuk and L. T. Pedersen for technical assistance, and J. Juul and J. Karlsen for key support on molecular animations. Support for this work was provided by grants from the NIH to S.C.B. (grant number 1R01GM098859), by an EMBO Short-Term Fellowship to M.D., and by Lundbeck Foundation and AIAS COFUND fellowships to M.K. We also thank the Molecular Machines COST Action (CM1306) for helpful support.
S.C.B. and R.B.A. have an equity interest in Lumidyne Technologies.
Reviewer Information Nature thanks G. Bultnyck, S. Karlish and A. van Oijen for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Figure 1 Functional cycle of P-type ATPases illustrated with SERCA crystal structures.
SERCA crystal structures (PDB accession numbers in clockwise order starting from E1: 4H1W6, 1T5S2, 1T5T2, 3B9B5, 3B9R5, 2C884). The distances between LMCA1TM–A/P labelling sites (T24C and A530C, corresponding to SERCA P26 and E606; shown as green spheres connected by a black line) are shown. The N, P and A domains are shown as red, blue and yellow cartoons, respectively. Transmembrane helices M1–M2, M3–M4 and M5–M10 are shown as pink, wheat, and grey cartoons, respectively. Ca2+ ions are shown as orange spheres. Sarcolipin associated with the 4H1W crystal structure is shown as a cyan cartoon representation. Mutations used to inhibit or slow down partial reactions are depicted in red. Square brackets denote ion occlusion. Ion stoichiometry in the scheme refers to LMCA1.
a, Activity measurements were performed under conditions resembling those used for smFRET experiments (1 mM Ca2+, 1 mM ATP, 10 mM Mg2+ and pH 7.6). Time course measurements (n = 2 technical replicates) were subjected to linear regression; the slope provided a measure of the activity. Error bars correspond to 95% confidence intervals of the fit. b, 4.5 nmol of LMCA1TM–A/P was labelled with various amounts of Cy3 and Cy5 dyes (0, 22.5, 45, 90 nmol) in a final volume of 286 μl for 30 min. Labelling efficiency was defined as a corrected ratio of Cy3 or Cy5 absorbance to LMCA1TM–A/P absorbance. Horizontal error bars show s.d. of n = 2 labelling reactions with Cy3 or Cy5 dyes. Activity measurements were performed and analysed as described above.
a, Schematic of the surface immobilization and imaging strategy. Fluorophores (green sticks) are attached to the engineered cysteine labelling sites in LMCA1TM–A/P, which is depicted as a cartoon with A (yellow), N (red), P (blue), and TM (wheat) domains. b, Wide-field fluorescence images of surface-immobilized LMCA1TM–A/P particles. N is the total number of particles detected. c, Image taken after treatment of the surface with 0.3 M imidazole.
a, FRET values from all traces were summed into population histograms to reveal subtle shifts in mean FRET efficiency across conditions: 0.1 mM EGTA (black), 1 mM Ca2+ (orange), 1 mM Ca2+ with 1 mM ATP (purple), 1 mM Ca2+ with 1 mM ATPγS (green), and 0.1 mM EGTA with 0.2 mM BeFx (blue). Representative single molecule fluorescence (donor in green and acceptor in red) traces of LMCA1TM–A/P in 0.1 mM EGTA (b) and 1 mM Ca2+ (c). d, Mean FRET efficiency from experiments in the presence of the indicated concentration of Ca2+ and buffer at pH 7.5 (orange) or 6.0 (blue), fitted to Hill equations with EC50 values of 250 μM and 2.5 mM, respectively (vertical lines). The graph shows mean ± s.e.m., n = 3. e–h, Representative traces in: EGTA and BeFx (e); 1 mM Ca2+ and 1 mM ATP (f); 1 mM Ca2+ and 1 mM ATPγS (g); and 1 mM each of Ca2+, ADP and AlFx (h). i, j, Distribution of dwell times within the high-FRET (i) and low-FRET (j) states in the presence of 1 mM Ca2+ and 1 mM ATP imaged at 5 ms time resolution. Graph shows mean ± s.e.m., n = 5.
a, Representative trace of D334N-LMCA1TM–A/P in the presence of 10 mM Ca2+ and 1 mM ATP. Compare to Fig. 1h and Extended Data Fig. 4f. b, Population average FRET efficiency from representative experiments starting in 0.1 mM EGTA, followed by rapid delivery (vertical dotted line) of 10 mM Ca2+ and 1 mM ATP (red), 10 mM Ca2+ (black), 1 mM ATP with Mg2+ (light green) or 1 mM ATP without Mg2+ (dark green). Experiments were also performed starting in 1 mM ATP with subsequent addition of 10 mM Ca2+ (orange) and starting in 10 mM Ca2+ with subsequent addition of 1 mM ATP (purple). Values were normalized to the initial FRET value before injection. Lines are fits to exponential functions and represent a lower bound for the rate of ligand binding and the subsequent conformational change, limited both by finite mixing time and the time resolution of imaging. c, Time constants from the curves in b. Bars show mean of n = 2 repeats (squares). The ATP/Ca+ATP and Ca/Ca+ATP experiments have higher uncertainty owing to the smaller magnitude change. A response time on the order of a single 15 ms frame (horizontal dotted line) is consistent with the data in all cases.
Extended Data Figure 6 [Ca]E1P–ADP intermediate dwells before E2P formation in E167Q-LMCA1TM–A/P are rare and transient.
a, Example trace of E167Q-LMCA1TM–A/P imaged in EGTA, followed by rapid exchange with 1 mM ATP and 10 μM Ca2+ (vertical dotted line). The expected FRET value of Ca2+-bound states is indicated by a horizontal dotted green line. b, Traces were summed into a population contour plot, showing accumulation in low FRET over time after injection of substrates (vertical dotted line). c, Traces from b were individually post-synchronized to the transition to the low-FRET E2P state (vertical dotted line). No accumulation in higher-FRET states is apparent before the transition. Such accumulation would appear as increased density at FRET = 0.75 and reduced density at lower FRET values just before the vertical dotted line. d, E167Q-LMCA1TM–A/P was pre-incubated with EGTA and 1 mM ATP. The graph shows occupancy in the low-FRET E2P state following addition of Ca2+ and ATP (vertical dotted line) without (black) and with (red) 1 mM ADP. Lines are fits to exponential functions with time constants of 0.16 ± 0.02 and 0.18 ± 0.01, respectively. Points show mean ± s.d., n = 3.
a, E167Q-LMCA1WT and LMCA1WT preincubated with Ca2+ were phosphorylated for 10 s at 25 °C in a reaction buffer containing 1 mM CaCl2 and the indicated concentrations of ATP, followed by acid quenching. The solid lines show the best fits to the Hill equation with the Hill coefficient constrained to 1.0. The data are results from a single experiment with half of the points performed in duplicate. b, Quench–flow experiments were carried out at 25 °C by mixing E167Q-LMCA1WT pre-incubated in a reaction buffer supplemented with 0.2 mM EGTA with an equal volume of the same buffer supplemented with 2.2 mM CaCl2 and 10 μM [γ-32P]ATP (final concentration, ~1 mM CaCl2, 5 μM ATP), followed by acid quenching at the indicated times. Phosphorylation is shown relative to the maximum level reached (mean ± s.d., n = 2 independent experiments). The solid line is the best fit of a mono-exponential function. c, Quench–flow experiments were performed at 25 °C by mixing labelled E167Q-LMCA1TM–A/P pre-incubated in a reaction buffer containing 1 mM CaCl2 with an equal volume of the same buffer containing different concentrations of [γ-32P]ATP. Phosphorylation rates were calculated and plotted as a function of [γ-32P]ATP concentration. Error bars show 95% confidence intervals of the fit. The solid line shows the Michaelis–Menten equation resulting in the listed values of Vmax and KM.
a, Population FRET histograms from imaging G4-LMCA1TM–A/P in the presence of 0.1 mM EGTA (black); 1 mM Ca2+ (orange); 1 mM Ca2+ with 1 mM ATP (purple); 1 mM Ca2+ with 1 mM ATPγS (green); and 0.1 mM EGTA with 0.2 mM BeFx (cyan). b–d, Representative smFRET traces from experiments in the presence of EGTA, followed by exchange (vertical line) to 1 mM Ca2+ (b); 1 mM Ca2+ and 1 mM ATPγS (c); and Ca2+ and ATP (d) at low illumination intensity and 200 ms time resolution to show the behaviour of a single transporter molecule on the timescale of minutes. e, Low-FRET state occupancy of G4-LMCA1TM–A/P imaged in the presence of varying concentrations of ATP (purple), Ca2+ (orange), or Mg2+ (red) at pH 7.5; or varying pH (f). Lines are fits to dose–response functions. Graphs show mean ± s.e.m., n = 3. Unless specified, the concentrations of ATP, Ca2+ and Mg2+ are 1 mM, 1 mM and 10 mM, respectively. g, h, Population FRET–time contour plot (g) and representative FRET trace (h) from experiments starting in 1 mM Ca2+ and 1 mM ATP, followed by buffer exchange to replace ATP with 1 mM ATPγS. i, Population average FRET over time from experiments where ATP is replaced with ATPγS (green), ATP is removed (purple) or Ca2+ is removed (orange). Lines are fits to exponential functions with mean time constants of 380 ms. j, Mean frequency of transitions in the presence of 1 mM Ca2+, 1 mM ATP, and the indicated concentrations of ADP. The graph shows mean ± s.d., n = 2.
Extended Data Figure 9 Dwell time distributions of G4-LMCA1TM–A/P with varying ligand concentrations and pH.
G4-LMCA1TM–A/P was imaged in 1 mM ATP, 1 mM Ca2+ and 10 mM Mg2+ at pH 7.5 unless specified otherwise. Sampling of low- and high-FRET states was idealized as shown in Fig. 3b. Graphs show the distribution of dwell times in the high-FRET (a–d) and low-FRET (e–h) states; dwell counts were normalized to the imaging time before photobleaching. Experiments were performed varying the concentration of ATP (a, e); Ca2+ (b, f); or Mg2+ (c, g). In each case, the concentration of the ligand varied as: zero (0.1 mM EGTA; black), 1 μM (blue), 10 μM (green), 100 μM (orange), 1 mM (red) and 10 mM (dark red). d, h Varying pH: 6.0 (black), 6.5 (blue), 7.0 (green), 7.5 (orange), 8.0 (red) and 8.5 (dark red). Error bars are mean ± s.e.m., n = 3.
Extended Data Figure 10 G4-E167Q-LMCA1TM–A/P transits a [Ca]E2P–ADP intermediate on the path to Ca2+ site de-occlusion and stable E2P formation.
G4-E167Q-LMCA1TM–A/P was pre-incubated in 1 mM Ca2+, followed by addition of 1 mM ATP (time zero). a, Representative single-molecule fluorescence and FRET traces from this experiment, with [Ca]E1–ATP, [Ca]E2P–ADP and E2P phases highlighted in yellow, red and green, respectively. b, c, Ensemble FRET histograms of raw traces (b) and traces synchronized to the drop in FRET associated with [Ca]E2P−ADP state formation (c).
This file contains Supplementary Table 1 (distance changes expected from crystal structure models) and Supplementary Table 2 (dose-response fit parameters). (PDF 133 kb)
The observed dynamics of the LMCA1 cycle have been applied and scaled to a morphed series of conformational changes in rabbit SERCA1a based on the following PDB accession numbers (functional states): 4H1W (E1), 1T5S ((Ca)E1-ATP), 1T5T ([Ca]E1P-ADP), 3B9B (E2P), 3B9R ([H]E2P dephosphorylation), 2C88 ([H]E2). The relative transition rates applied to the morph were based on the data summarized in Fig. 4. (MOV 8840 kb)
About this article
Cite this article
Dyla, M., Terry, D., Kjaergaard, M. et al. Dynamics of P-type ATPase transport revealed by single-molecule FRET. Nature 551, 346–351 (2017). https://doi.org/10.1038/nature24296
The Journal of Membrane Biology (2021)
Journal of Fluorescence (2020)
Biophysics Reports (2019)