AbstractPlasma-membrane Ca2+-ATPases expel Ca2+ from the cytoplasm and are key regulators of Ca2+ homeostasis in eukaryotes. They are autoinhibited under low Ca2+ concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases the autoinhibition and activates the pump. However, the structural basis for this activation, including the overall structure of this calcium pump and its complex with calmodulin, is unknown. We previously determined the high-resolution structure of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8 and revealed a bimodular mechanism of calcium control in eukaryotes. Here we show that activation of ACA8 by CaM involves large conformational changes. Combining advanced modeling of neutron scattering data acquired from stealth nanodiscs and native mass spectrometry with detailed dissection of binding constants, we present a structural model for the full-length ACA8 Ca2+ pump in its calmodulin-activated state illustrating a displacement of the regulatory domain from the core enzyme. IntroductionCalcium ions (Ca2+) play a major role as secondary messengers in numerous signal transduction processes (reviewed in the ref. 1,2). In order to maintain the concentration gradient between low intracellular (100鈥塶M) and high extracellular (2鈥塵M) Ca2+ that sets the stage for calcium signaling1, eukaryotic cells have evolved a sophisticated regulation system involving plasma-membrane Ca2+-ATPases (PMCAs). These high-affinity Ca2+ pumps export Ca2+ ions from the cytosol into the extracellular environment using energy provided through ATP hydrolysis, and are tightly regulated. They help to maintain overall Ca2+ homeostasis and provide local control of intracellular Ca2+ signaling3,4.PMCAs belong to the P2B subfamily of P-type ATPases5 and play a major physiological role in e.g., for pre-synaptic and post-synaptic Ca2+ regulation in neurons, feedback signaling in the heart and sperm motility6. Compared to other P-type ATPases, plasma-membrane calcium ATPases contain an additional autoinhibitory or regulatory domain. While mammalian PMCAs contain a C-terminal autoinhibitory domain, in plant ACAs (autoinhibited Ca2+-ATPases) this domain is located at the N-terminus7. Binding of calmodulin (Ca2+-CaM) to this region relieves autoinhibition and results in pump activation, however, the conformational changes leading to PMCA activation are not well understood.To investigate the structural basis for PMCA activation, we have dissected the underlying binding events of autoinhibition and activation using biochemical and biophysical methods, and combined this analysis with small-angle neutron and X-ray scattering studies of a PMCA pump reconstituted in stealth nanodiscs, facilitating its structural characterization in a lipidic solution environment. Using this hybrid approach, we developed a structural model for the activation of plasma-membrane Ca2+-ATPases by calmodulin. In its calmodulin-activated state, the regulatory domain is displaced from the core, enabling maximal enzymatic activity. The combination of stealth nanodiscs, selectively deuterated protein components and SANS should be broadly applicable to many membrane protein complexes.ResultsBinding of two calmodulin molecules to ACA8 leads to its activationFull-length ACA8 protein was expressed and purified to homogeneity and its state of inhibition was assessed by the basal ATPase activity. Binding of calmodulin (CaM7, hereafter denoted as CaM) to the CaM-binding domain (CaMBD) of detergent solubilized ACA8 leads to a two-fold increase in ATPase activity of the pump compared to the basal activity in the absence of CaM (Fig.聽1a). To gain insights into the stoichiometry of the ACA8-CaM complexes, DDM-solubilized and purified ACA8 in its apo and CaM-bound states was analyzed by native mass spectrometry. Native mass spectrometry is an established method for the precise determination of protein stoichiometries in soluble and membrane protein complexes8. The spectrum shows peaks corresponding to the apo ACA8, ACA8-CaM (single CaM) and ACA8-CaM2 (two CaM molecules bound) (Fig.聽1b). In the full m/z range spectrum highly charged CaM is detected, which dissociated from the weakly charged ACA8 and the ACA8-CaM complex (Supplementary Fig.聽1). These findings show that a maximum of two CaM molecules can bind to the full-length ACA8 as previously observed for the isolated regulatory domain and thereby confirm the proposed two-step activation mechanism9.Fig. 1Incorporation of ACA8 in nanodiscs and activation by CaM-binding. a ACA8 ATPase activity assay. Activity was measured (in triplicates) in detergent micelles and nanodiscs containing different lipids in absence or presence of calmodulin. b Native mass spectrometry showing up to two CaM molecules bound to full-length ACA8 in detergent solubilized state. At high acceleration voltages (200鈥塚) proteins are released from the detergent micelles. The masses (Supplementary Table聽1) indicate the presence of unbound ACA8 (black), ACA8-CaM (light blue) and ACA8-(CaM)2 (dark blue). c CaM-dependent activity measurements of nanodisc-incorporated ACA8. Measurements were performed in nanodiscs containing POPC (black) or POPC/soyPI mixture (gray) as lipid component with different amounts of CaM added. d Size-exclusion chromatography profile of ACA8 in nanodisc (black line) and in complex with CaM (blue line) showing an expansion once CaM is boundFull size imageIn order to investigate the structure and conformational changes of ACA8 in a native-like lipidic environment, we reconstituted the pump in MSP1D1 nanodiscs (ND) composed of various lipids (Supplementary Fig.聽2). Size-exclusion chromatography profiles of ACA8 incorporated in nanodiscs for all different types of lipids indicated a monodisperse sample preparation (Fig.聽1d). ATP hydrolysis assays in different lipid reconstitutions revealed much higher activity of CaM-activated ACA8 in the nanodiscs composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) compared to the activity of the ACA8-CaM complex in detergent micelles (Fig.聽1a), emphasizing the importance of a lipidic environment for the proper function and activation of ACA8 as previously demonstrated for human PMCAs10,11. Reconstitution of ACA8 into nanodiscs containing 70% POPC and 30% POPG or 70% POPC and 30% soy-PI results in a further 3鈥?-fold activity increase of CaM-activated ACA8 compared with POPC alone, showing that negatively charged head groups are able to further increase the enzymatic activity once the pump is activated (Fig.聽1a). Overall, our absolute activity values as well as the activation factors (activity of ACA8鈥?鈥塁aM/activity of ACA8) for nanodisc-incorporated samples are in good agreement with previous results obtained using microsome preparations12,13,14,15. To further improve聽our understanding of the role of lipids in ACA8 activation by CaM we analyzed the basal ATPase activity of ACA8 by titrating CaM to the apo-ACA8 in POPC and POPC/soy-PI nanodiscs. In both instances, we observe concentration-dependent increase in the ATP hydrolysis rate, revealing two transitions, which reflect the proposed bimodular activation mechanism9 (Fig.聽1c). Additionally, these experiments show a marked effect of ACA8 activation by negatively charged lipids at higher CaM concentrations. Above a CaM concentration of approx. 500鈥塶M, the activity of ACA8 in nanodiscs composed of soy-PI is shifted to higher pump rates, indicating that once two CaM molecules are bound, the anionic headgroup of soy-PI further stimulates the activity. These findings are in good agreement with previous studies on PMCAs with a photo-activatable phosphatidylcholine analog, showing that CaM and phosphatidic acid behave independently regarding their effects on enzyme activity and transmembrane conformation16. Altogether, these results support the bimodular activation mechanism and further show the importance of diverse surrounding lipids for the activation process.CaM-binding induces large conformational changes in ACA8In order to investigate the structural changes leading to the activation of ACA8 by CaM, we carefully dissected the different binding affinities of the various protein-protein interactions during activation by using fluorescence anisotropy (Fig.聽2). Fluorescence anisotropy detects differences in rotational speed, which in turn correlates with differences in molecular masses (if the mass differences are large enough). All experiments were performed using ACA8 constructs reconstituted in POPC-nanodiscs.Fig. 2Binding affinities within ACA8-CaM complex investigated by fluorescence anisotropy. a Binding of fluorescein-labeled CaM to full-length ACA8 (Kd鈥?鈥?8鈥塶M). b Binding of fluorescein-labeled regulatory domain (RD) to ACA8core (Kd鈥?鈥?.7鈥壩糓). c Addition of CaM to a preformed complex containing ACA8core and labeled RD. Decrease in anisotropy indicates displacement of RD from ACA8core through CaM-binding to RD. All experiments were performed using protein reconstituted in nanodiscsFull size imageWe first measured binding of fluorescein-labeled CaM to full-length ACA8, revealing tight binding with an apparent Kd of 18鈥塶M (Fig.聽2a). In this setup, the measured affinity corresponds to binding of CaM to the high-affinity binding site and resembles the value previously determined for CaM-binding to the isolated regulatory domain9. To characterize the structural changes during the activation we expressed and purified the regulatory domain of ACA8 (ACA8RD, residues 1鈥?30) alone in E. coli. Next, we assessed the level of secondary structure in ACA8RD using far-UV circular dichroism spectroscopy. These experiments indicated that this domain consists partly of random coil segments with some degree of 伪-helicity, presumably spanning residues 40鈥?5 as observed in the ACA8RD-(CaM)2 crystal structure9 (pdb:4aqr) (Supplementary Fig.聽3). Titration of the fluorescein-labeled ACA8RD to the core of ACA8 (residues 131鈥?074, a construct lacking the regulatory domain) yields a dissociation constant (Kd) of 1.7鈥壩糓 (Fig.聽2b). Addition of CaM to this preformed complex resulted in a decrease in anisotropy (K0.5 approx. 120鈥塶M), indicating that the displacement of ACA8RD from the core of ACA8 is dependent on CaM-binding to ACA8RD (Fig.聽2c). The displacement setup described in Fig.聽2c reports indirectly on the binding of the second CaM molecule to the low-affinity binding site, as the regulatory domain will be only displaced from the ACA8core construct once both CaM are bound. Overall, these binding experiments indicate that the regulatory domain binds weakly to the core of the ACA8 pump. Binding of two CaM molecules to regions on the regulatory domain that are also involved in binding to the catalytic core12 can then displace the regulatory domain from the core enzyme. The fact that the regulatory domain has a higher affinity to CaM than to the core enzyme suggests that the availability of Ca2+-loaded CaM is the dominant factor in the regulation of pump activity.Displacement of the regulatory domain caused by CaM-binding can also be observed by size-exclusion chromatography (SEC). The SEC-profile of the ACA8鈥?鈥塁aM complex in ND reveals a shift of the elution peak (11.1鈥塵l for complex vs. 11.9鈥塵l for ACA8 alone) that cannot be simply explained by the binding of CaM but rather indicates a hydrodynamic expansion of the complex in solution leading to this drastic change in the particle elution volume (Fig.聽1d).With these data in hand the structural changes during activation were further studied by small-angle X-ray scattering (SAXS). SAXS is a suitable technique to determine overall dimensions and shapes as well as flexibility and larger conformational changes of biomolecules in solution. We performed SAXS measurements of POPC-nanodisc-incorporated ACA8 and its complex with CaM (Fig.聽3). The calculated Mr from I(0) values are in excellent agreement with the theoretical values (Table聽1). Binding of CaM leads to an increase in radius of gyration (Rg) from 5.3鈥塶m for the apo ACA8 to 5.88鈥塶m for the CaM-activated state and a slight increase in maximum diameter (Dmax) (20鈥塶m/22鈥塶m) once CaM is bound (Table聽1/Supplementary Fig.聽4). Beside the increase in Rg and Dmax the dimensionless Kratky plot ((qRg)2 x I(q)/I(0) vs. qRg)聽 shows differences in the mid q-range where usually domain movements are visible, indicating conformational rearrangement and increased flexibility, which is most likely due to the displacement of the regulatory domain upon CaM binding (Fig.聽3b). This data is in line with the fluorescence anisotropy data and shows that the conformational changes upon activation of ACA8 by CaM can be detected by small-angle X-ray scattering. Moreover, we analyzed the flexibility of the regulatory domain in the activated state using the Porod-Debye plot. A plot of q4I(q) vs. q4 will achieve a plateau for more compact molecules and not for more flexible molecules. The ACA8-CaM complex does not show a plateau in the low q-range of the Porod-Debye plot (q4鈥?lt;鈥?.1鈥塶m鈭?), indicative of a more diffuse contrast that is consistent with a flexible conformation. A clear plateau is visible for ACA8 in its autoinhibited state (Fig.聽3c). SAXS measurements with ACA8 incorporated in nanodiscs containing anionic lipids (POPG or soy-PI) did not show any expansion in the absence of CaM with radii comparable to those of ACA8 in POPC-nanodiscs (Supplementary Fig.聽5). Binding of CaM to ACA8 in nanodiscs containing anionic lipids leads to an increase in Rg. This indicates that anionic lipids are not sufficient to fully activate the pump on their own, however they seem to play a role in later stages of CaM activation. We speculate that anionic lipids might stabilize an intermediate conformation that can readily be activated by CaM-binding leading to high activity gain (Fig.聽1a).Fig. 3Small-angle X-ray scattering (SAXS) analysis of nanodisc-incorporated ACA8 and its complex with CaM. a Binding of CaM to ACA8 leads to an increase in Rg and Dmax. b Dimensionless Kratky plot of the data in a highlighting the conformational change of the regulatory domain upon CaM binding to ACA8. c Porod-Debye-Plot plot without plateau for the ACA8-CaM complex indicating a decrease in the overall contrast due to a more flexible conformation. Data for ACA8 in ND are depicted in black, data for the corresponding complex with CaM are shown in blueFull size imageTable 1 SAXS parametersFull size tableStructural model of activated ACA8-(CaM)2 complex in nanodiscsIn order to investigate the structure of ACA8 in its autoinhibited as well as CaM-activated state in a native-like lipid environment we made use of the recently developed stealth nanodisc (sND) technology (Fig.聽4d)17,18. Stealth nanodiscs are fractionally deuterium labeled scaffolds consisting of phosphatidylcholine lipids and MSP1D1 belt protein; these can be rendered effectively invisible to low-resolution neutron diffraction using the appropriate solvent contrast and allow the low-resolution structure determination of integral membrane proteins in a lipid environment without contribution of the scaffold to the small-angle neutron scattering (SANS) signal (Fig.聽4d). Using this stealth nanodisc / SANS method structural changes involving the incorporated membrane protein are usually easier to detect and to model compared to SAXS experiments, as in the sND/SANS setup only the components of interest contribute to the scattering signal, while in the SAXS setup the nanodisc also contributes to the signal. The SAXS measurements, however, provide important independent controls, as the overall structural trends (such as increase in Rg, Dmax, flexibility etc.) should also be observable.Fig. 4Small-angle neutron scattering (SANS) analysis of ACA8 and its ACA8-CaM complexes in stealth carrier nanodiscs. a Comparison of SANS scattering data of ACA8 in sND in 100% D2O in apo conformation (black) with data for the corresponding complex with hydrogenated (hCaM) and deuterated CaM (dCaM) (light blue and dark blue, respectively). The deuterated nanodisc components do not contribute to the SANS scattering signal in 100% D2O18. Scattering signal for deuterated CaM (73% deuterated) is shown in gray, showing that dCaM is fully matched out. b Distance distribution (P(r))-plot of the data shown in a indicating that binding of CaM to ACA8 leads to an increase of Dmax. c Activity assay in absence and presence of CaM. d Cartoon illustration of the used stealth carrier nanodisc technique. At 100% D2O the nanodisc components are fully matched out (shown in semi-transparent gray) and only ACA8 (violet) contributes to the scattering signalFull size imageControl experiments confirmed that the deuterated disc constituents do not affect the activation process of ACA8 by CaM, as the expansion in hydrodynamic radius as well as the activity is comparable to those in non-deuterated nanodiscs (Fig.聽4c; Table聽2). SANS data were acquired (all in sND) (1) for apo-ACA8, (2) ACA8 in complex with protonated CaM, and (3) ACA8 in complex with perdeuterated CaM (dCaM), where only ACA8 in its activated state contributes to the scattering signal as the contrast matched-out CaM is invisible to neutron scattering (Fig.聽4). From the SANS scattering data we could calculate parameters of overall size (Rg, Dmax) that clearly show expansion of ACA8 upon CaM-binding (Table聽2/Supplementary Fig.聽6).Table 2 SANS parametersFull size tableThe experimentally derived radius of gyration for ACA8 in stealth nanodiscs was 4.0鈥塶m for the apo conformation, and the distance distribution function has a bi-lobed shape with two peaks, at 3.6 and 7.0鈥塶m, and a maximal particle dimension of 13鈥塶m, which is consistent with a rather compact multi-domain particle (Fig.聽4b). Once CaM binds to ACA8 both Rg and Dmax show a large increase to 4.3鈥塶m and 15鈥塶m, respectively, for the complex with deuterated CaM, which is in line with the SAXS data. The complex of ACA8 with hydrogenated CaM shows an even bigger Rg and Dmax of 5.0鈥塶m and 18鈥塶m, respectively, indicating that the deuterated CaM is effectively matched out in the ACA8-sND鈥?鈥塪CaM complex (Fig.聽4a, b; Table聽2). The peak at 7.0鈥塶m in the distance distribution function becomes less pronounced and shifts towards larger distances when either hydrogenated or deuterated CaM are used, indicative of protein expansion. These data are consistent with the model that the binding of the regulatory domain to the core is released and the ACA8 structure becomes less compact relative to the apo conformation (Fig.聽4b), as already indicated by the SAXS experiments. Notably, all Rg values derived from SANS measurements in sND are smaller than those obtained by SAXS indicating the minimal scattering contribution of the nanodisc to the SANS data (Tables聽1 and 2). For this reason, the observed changes upon activation by CaM are more pronounced in our SANS data using sND compared to the SAXS data.In order to generate a structural model for ACA8 in its activated CaM-bound state, high-quality ACA8鈥?鈥塪CaM stealth nanodisc data were obtained from SANS measurements. With the activating CaM and lipid nanodiscs rendered invisible due to the partial deuteration strategy employed, the acquired dataset represents a fully-activated ACA8 in a native-like lipid environment. We generated a homology model of the 鈥淎CA8 core鈥?(residues 130鈥?074) based on a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) structure in E2 conformation (pdb:3b9b). We used the long helix (residues 40鈥?5) from the previously determined crystal structure of the ACA8 regulatory domain in complex with CaM9 (pdb:4aqr) and generated models of the linker (96鈥?30) and the N-terminal extension (1鈥?0) in various conformations using the program RANCH19,20. The quality of these models was evaluated by back-calculating the scattering intensities from the models and fitting against the measured SANS data of the complex with deuterated CaM (Fig.聽5). Although the overall size and shape of the homology model complexes describe the low-q regions of the SANS data well, no single model was found to provide a satisfactory fit to聽the experimental data across the entire data range. As the regulatory domain is expected to be more flexible in the activated state we performed ensemble optimized modeling (EOM) analysis to quantitatively assess changes in the ensemble-averaged conformation upon CaM binding. The use of EOM for the modeling of SANS data acquired for integral membrane proteins in stealth nanodiscs is a very promising strategy for the structural characterization of flexible IMPs. EOM uses a genetic algorithm to select an ensemble of conformers of the regulatory domain whose weighted averaged scattering curve best reproduces the experimental scattering data19,20. The selected ensemble (best performing sub-ensemble) reveals an excellent fit to the experimental data (chi2鈥?鈥?.0) (Fig.聽5b). Three representative models with Rg values between 4.0 and 4.4鈥塶m show different orientations of the regulatory domain (Fig.聽5c) and as an ensemble describe the conformational flexibility of the complex. None of the selected conformers contains a fully extended regulatory domain, which is also apparent in the Rg distribution plot that is shifted to smaller Rg values for the selected pool compared to the random pool. The Rg size-distribution indicates that the complex occupies a restricted range of conformations that tend towards the more compact rather than extended conformations, relative to the random pool. This is also reflected in the metric Rflex that provides a quantitative measure for the flexibility of the system, with Rflex being 68.1% for the selected ensemble and 86.1% for the pool.Fig. 5Structural model of the activated ACA8-(CaM)2 complex. The Ensemble Optimization Method (EOM)20 was used to generate and select a pool of models with different conformations of the regulatory domain (a) that were subsequently used to fit the experimental SANS data (acquired in stealth nanodiscs) (b). c鈥?b>h The resulting representative models for the activated ACA8-(CaM)2 complex from the best-fitting sub-ensemble show ACA8core (gray) with the regulatory domain in three different conformations (cyan, green and orange). f鈥?b>h are rotated by 90 degreesFull size imageConclusionsTaken together, these data illustrate that CaM-binding to ACA8 leads to displacement of the regulatory domain from the ACA8 core enzyme. This displacement of the regulatory domain releases the autoinhibition and allows the enzyme to undergo its conformational cycle required for the pumping of Ca2+ ions. In combination with our previous crystal structure of the ACA8 regulatory domain in complex with CaM9, this structural model for activated ACA8 reveals the structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin in the context of the full-length protein in a native-like lipid environment. The results add further structural insights into the previously proposed bimodular activation mechanism9 showing that binding of two CaM molecules leads to full activation of this Ca2+ pump via displacement of the regulatory domain.MethodsMaterialsDetergents and lipids were purchased from the following companies: DDM (#D310/Anatrace), LMNG (#NG310/Anatrace), cholate (#3407.1/Roth), POPC (#850457鈥塒/Avanti Polar Lipids), POPG (#840457P/Avanti Polar Lipids), soyPI (#840044P/Avanti Polar Lipids). All other chemicals were of analytical grade and obtained from Roth (Karlsruhe, Germany) or Sigma Aldrich (St. Louis, MO, USA).Expression and purification of full-length ACA8, ACA8core, and ACA8RDACA8 was overexpressed with an N-terminal His8-tag in S. cerevisiae strain BJ5460 (MATa ura352 trp1 lys2801 leu2delta1 his3delta200 pep4::HIS3 prb1delta1.6R can1 GAL)21 using a pYES2 plasmid. Large-scale cultures were grown in uracil-depleted media (6.7鈥塯鈥塴鈭? YNB)鈥?鈥?.1% glucose at 30鈥壜癈 up to an OD600 of 0.6 and expression was induced by adding 2% galactose. Cells were harvested 20鈥塰 post induction by centrifugation at 3000xg and resuspended in 30鈥塵M Tris pH 8.0, 300鈥塵M NaCl, 20% (v/v) glycerol, 3鈥塵M 脽-mercaptoethanol, 20鈥塵M EDTA (buffer A) before being disrupted with glass beads. Following clearing of the lysate, cell membranes were isolated by centrifugation at 180,000脳g and membranes were solubilized in buffer A with 1% lauryl maltose neopentyl glycol (LMNG) for 1.5鈥塰 with gentle stirring. To remove insoluble material the solubilized membranes were centrifuged at 100,000脳g and supernatant were incubated with Ni2+ affinity resin. Resin was washed with buffer containing 30鈥塵M Tris pH 8.0, 300鈥塵M NaCl, 2鈥塵M CaCl2, 1鈥塵M 脽-mercaptoethanol, 0.005% LMNG and 40鈥塵M imidazole and ACA8 were eluted with 150鈥塵M imidazole. The purity of ACA8 was judged by SDS-PAGE and corresponding fractions were pooled and concentrated to 2鈥塵g/ml before reconstituted into nanodiscs. The ACA8 mutant lacking the regulatory domain (ACA8core) was expressed and purified in the same way as the full-length protein.The regulatory domain of ACA8 (aa 1鈥?30) (ACA8RD) was cloned as a fusion construct with an N-terminal His6-lipoamyl-TEV-tag22 in the pET28a vector and a cysteine was introduced right before the first residue of the regulatory domain to allow site specific labeling with thiol-reactive probes. ACA8RD was co-expressed together with CaM7 in E. coli strain C4123, grown in 2xTY media at 20鈥壜癈 for 16鈥塰. Cells were harvested at 4000脳g and resuspended in buffer containing 30鈥塵M Tris pH 7.5, 300鈥塵M NaCl, 10% glycerol, 2鈥塵M CaCl2, 1鈥塵M Tris-(2-carboxyethyl)-phosphin (TCEP) (buffer D) before lysed using high-pressure homogenizer (EmulsiFlex-C3, Avestin). The cleared lysate was loaded on HisTrap column, pre-equilibrated with buffer D and washed with 20鈥塵M imidazole before eluted with 200鈥塵M imidazole followed by TEV protease digestion and a second nickel affinity chromatography step to separate the His-lipoamyl-TEV tag.Expression and purification of CaM7CaM7 from A. thaliana was used as calmodulin ortholog throughout this study. CaM7 in pET42a vector was transformed into E.coli Bl21 Gold (DE3) and grown in LB medium at 37鈥壜癈. After induction of protein expression with 0.5鈥塵M isopopropyl 脽-D-1-thiogalactopyranoside (IPTG), cells were grown for another 16鈥塰 at 20鈥壜癈 and harvested by centrifugation at 3000 脳 g. Cells were resuspended in 30鈥塵M Tris pH 7.5, 50鈥塵M NaCl, 1鈥塵M 脽-ME, 2鈥塵M CaCl2 (buffer B) and broken using high-pressure homogenizer (EmulsiFlex-C3, Avestin). The lysate was cleared by centrifugation at 40,000 脳 g and the supernatant was bound to HiTrap Phenyl HP column, pre-equilibrated with buffer B. CaM7 was eluted with 5鈥塵M EDTA. Fractions containing pure CaM7 were pooled, concentrated to 10鈥塵g/ml and stored at 鈭?0鈥壜癈 until further use.Expression and purification of membrane scaffold protein MSP1D1MSP1D1 in pET28a vector was transformed in E.coli strain BL21 (DE3) and grown in terrific broth (TB) media at 37鈥壜癈. At an OD600 of 1.5 the protein expression was induced by adding 1鈥塵M isopropyl 脽-d-1-thiogalactopyranoside (IPTG) and cells were grown for 4鈥塰 at 37鈥壜癈. The protein was purified according to a modified protocol established by Sligar and co-workers24. Briefly, cells were harvested by centrifugation at 3000脳g, resuspended in lysis buffer (50鈥塵M Tris pH 8.0, 500鈥塵M NaCl) with 1% Triton X鈥?00 and broken using sonication. The cleared lysate was loaded onto a HisTrap column and washed with ten column volumes each of lysis buffer containing 1% Triton X-100 and 50鈥塵M cholate, respectively. MSP1D1 was eluted with buffer containing 500鈥塵M imidazole, and fractions containing pure protein were pooled and incubated with TEV protease overnight. Subsequently, the protease and cleaved His-tag were separated by applying a second IMAC chromatography step and MSP1D1 without His-tag was concentrated up to 400鈥壜礛 and stored at 鈭?0鈥壜癈 until further use.Expression and purification of deuterated membrane scaffold protein MSP1D1 (dMSP1D1), deuterated phosphatidylcholine (dPC), and deuterated calmodulin (d-CaM7)Matchout-labeled MSP1D1 (d-MSP1D1) was overexpressed in E. coli strain BL21 (DE3) as previously described25,17. After adaption of the strain to minimal deuterated medium26, cultures were grown in flaks in 85% deuterated minimal medium with glycerol as a carbon source27. The protein was purified according to a modified protocol established by Sligar and co-worker24 (as described above for non-deuterated protein).Selectively deuterated mixed acyl phosphatidylcholine, PC, was produced in a modified E. coli strain grown in minimal 100% deuterated medium supplemented with deuterated glycerol (C3D8O3) and partially deuterated choline chloride (trimethyl- d9, 98%; Eurisotop) as previously described28. Total phospholipids were extracted using the method of Bligh and Dyer29 and purified according to head-group using silica-gel column chromatography with varying ratios of chloroform and methanol as previously described28.Matchout-labeled CaM7 (d-CaM7) with a deuteration level of 73% was overexpressed in E. coli strain BL21 (DE3) adapted to growth in deuterated minimal medium30. A 1.8鈥塴 (final volume) deuterated high cell-density fed-batch fermenter culture25 was carried out at 30鈥?sup>慰C. Feeding with glycerol was started at an OD600 value of about 5. Expression of d-CaM was induced at an OD600 of about 12 by addition of IPTG (0.5鈥塵M final concentration). Cells were harvested at an OD600 of 19 yielding 100鈥塯 wet weight of matchout-labeled cell paste. Cells were resuspended into buffer containing 30鈥塵M Tris pH 8.0, 100鈥塵M NaCl, 1鈥塵M CaCl2, lysed using a high-pressure homogenizer (EmulsiFlex-C3) and purified by hydrophobic affinity chromatography (using a HiTrap Phenyl HP column), as described above.Reconstitution of ACA8 into (stealth) nanodiscsFor reconstitution of ACA8 or ACA8core into nanodiscs, 50鈥塵M lipids (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1鈥?rac-glycerol) (POPG) or L-伪-phosphatidylinositol (soy-PI) (Avanti Polar Lipids)) were dissolved in a buffer containing 30鈥塵M Tris pH 8.0, 200鈥塵M NaCl, 2鈥塵M CaCl2, 1鈥塵M 脽-mercaptoethanol (buffer C) and 100鈥塵M cholate. Lipid mixtures contained 70% POPC and 30% POPG or 70% POPC and 30% soy-PI, respectively. ACA8, the MSP1D1 membrane scaffold protein and the lipids were mixed in a molar ratio of 1:5:150 in buffer C containing 20鈥塵M cholate and incubated for 1鈥塰 at 4鈥壜癈. By adding detergent removal beads (Thermo Fischer Scientific) in a 1:1 (v/v) ratio, detergents were removed to initiate the nanodisc assembly and the sample was incubated over night at 4鈥壜癈 under constant agitation. Detergent removal beads were removed and the sample cleared by centrifugation before subsequent purification of nanodisc-embedded ACA8 on a Superdex200 column (GE Healthcare) in a buffer containing 20鈥塵M Tris pH 8.0, 150鈥塵M NaCl, 1鈥塵M CaCl2, 1鈥塵M 脽-mercaptoethanol. For reconstitution of ACA8 in stealth carrier nanodiscs (sND) the molar ratio of all components and the protocol was kept unchanged, but deuterated MSP1D1 and deuterated PC were used and the assembled stealth nanodiscs were dialyzed extensively against D2O-based buffer.Native mass spectrometry (NMS)Detergent (DDM)-solubilized ACA8 was exchanged to 200鈥塵M ammonium acetate solution pH 8.3, 2x CMC (0.018%) DDM using centrifugal filter units (Vivaspin 500, 100,000 MWCO, Sartorius) at 4鈥壜癈 and 15,000脳g. Nano electrospray ionization (ESI) capillaries were prepared as described before31.Native MS experiments were performed with a nanoESI source in positive ion mode on a QToF2 (Waters and MS Vision) that was modified for the analysis of high-mass ions32. 7 mbar source pressure and 1.7鈥壝椻€?0鈥? mbar argon as collision gas were used. Capillary and cone voltages were set to 1.7鈥塳V and 190鈥塚, respectively. The collision energy was ramped up to 400鈥塚, the illustrated spectra were recorded at 200鈥塚. CsI (25鈥塵g/ml) spectra were acquired and used to calibrate raw data using MassLynx software (Waters). Data were analyzed using MassLynx and Massign33.Activity measurementsACA8 activity was measured either in LMNG or nanodisc composed of different lipids as well as stealth carrier nanodisc using the Baginski assay34. All reactions were performed in buffer containing 150鈥塵M NaCl, 30鈥塵M Tris-HCl (pH 7.4 at 25鈥壜癈), 2鈥塵M MgCl2, 1.95鈥塵M EGTA, and 2鈥塵M CaCl2, resulting in 50鈥壩糓 final free Ca2+ concentration. Three microgram of purified ACA8 in LMNG and 2鈥壜礸 ACA8 in nanodisc were incubated in 50鈥壜祃 sample buffer with 1鈥塵M ATP for 10鈥塵in at 25鈥壜癈 before the reaction was stopped by adding 50鈥壜祃 ascorbic acid solution (140鈥塵M ascorbic acid, 0.5鈥塎 HCl, 0.1% SDS, 5鈥塵M ammonium heptamolybdate). The addition of 75鈥壜祃 containing 75鈥塵M sodium citrate, 2% (w/v) sodium metaarsenite and 2% (v/v) acetic acid stopped the colorimetric reaction and the absorbance at 860鈥塶m was read on a Tecan Infinite200 microplate reader after 10鈥塵in. Calmodulin was added in a range between 5鈥塶M and 10鈥壜礛 prior ATP addition. All activity measurements were performed under initial velocity conditions. The spontaneous and non-enzymatic hydrolysis of ATP just in buffer was subtracted from the measurements with ACA8. Mixed lipids contained 70% POPC and 30% POPG or 70% POPC and 30% soy-PI, respectively. A calibration curve using sodium phosphate in a concentration range from 0.01 to 0.6鈥塵M was used for determining the concentration of released phosphate. All reactions were measured as triplicates.Fluorescence anisotropy titrationFor fluorescence anisotropy measurements Alexa Fluor 488 C5 maleimide was used to label ACA8RD or CaM. For fluorescence labeling Alexa Fluor 488 C5 maleimide were added to 40鈥壜礛 ACA8RD-(CaM7)2 or 40鈥壜礛 CaM, respectively, in a molar ratio of 10:1 and incubated overnight at 4鈥壜癈. The reaction were stopped by adding 5鈥塵M 脽-mercaptoethanol, free fluorescein isothiocyanate (FITC) was separated with a PD10 column and the ACA8RD-(CaM)2 complex were dissociated by adding 10鈥塵M EDTA followed by binding of ACA8RD to a cation exchange chromatography column to separate both proteins. Fractions containing ACA8RD were pooled, concentrated and flash frozen in liquid nitrogen until further use. FITC-labeled CaM was used directly after PD10 column. Measurements were performed on an Agilent Cary Eclipse fluorescence spectrophotometer. Fluorescence anisotropy was measured with excitation at 480鈥塶m and emission at 520鈥塶m and slit width of 10鈥塶m. Each measurement was integrated over 5鈥塻 and the photomultiplier voltage was set to 700鈥塚. Reactions were carried out at 20鈥壜癈 in buffer containing 30鈥塵M Tris pH 8.0, 150鈥塵M NaCl, 2鈥塵M CaCl2, 0.5鈥塵M TCEP. In the binding experiments, 25鈥壜礛 ACA8core (in POPC nanodiscs) were titrated into 50鈥塶M fluorescein-labeled ACA8RD or full-length ACA8 (in POPC nanodiscs) was titrated into 15鈥塶M FITC-labeled CaM. Dissociation constants were obtained by fitting anisotropy data to the equation corresponding to a one-site binding model with robs鈥?鈥?i>r0鈥?鈥?螖r 脳 [P]) / (Kd鈥?鈥塠P)]Small-angle X-ray scattering (SAXS)18 SAXS was measured using the Bio-SAXS instrument P12 on the storage ring Petra III (DESY, Hamburg, Germany)35. The scattered intensity was recorded as a function of the scattering vector q with (left| q right|) =鈥? (pi) sin胃/位, using a wavelength of 0.124鈥塶m. All SAXS measurements were carried out at 10鈥壜癈 in 30鈥塵M Tris pH 7.5, 150鈥塵M NaCl, 2鈥塵M CaCl2, 2鈥塵M MgCl2 and 0.5鈥塵M TCEP at protein concentrations ranging from 1 to 8鈥塵g/ml with exposure times of 20鈥壝椻€?.05鈥塻. The average of the data was normalized and background subtracted using automatic procedures on the beamline36. Calibration of the scattering intensity into absolute units of cm鈭? was performed using the forward scattering intensity of bovine serum albumin. The radius of gyration was evaluated from the experimental SAXS pattern using the Guinier approximation and as well as from the entire scattering curve using the program GNOM37. The latter also provided the distance distribution function, p(r), and the maximal dimension, Dmax (see Table聽1).Small-angle neutron scattering (SANS)18 SANS data of ACA8 in the autoinhibited as well as in the activated state (with hydrogenated and deuterated calmodulin) were collected at the SANS-1 beamline at Forschungs-Neutronenquelle Heinz Maier-Leibnitz (FRMII) in Munich38. All measurements were performed in 100% D2O buffer (30鈥塵M Tris pH 7.5, 150鈥塵M NaCl, 1鈥塵M MgCl2, 1鈥塵M CaCl2) using a sample concentration of 3.0鈥?.5鈥塵g/ml at 10鈥壜癈. Measurements at FRMII were performed at 5鈥壝?wavelength with a sample-detector distance of 5.5鈥塵 (0.01鈥?lt;鈥?i>q鈥?lt;鈥?.23鈥壝?sup>鈭?) where the detector was moved by 404鈥塵m in direction perpendicular to the beam. Water reference sample (H2O), buffers, empty cell, the direct beam and the total absorber boron-cadmium were measured as well to perform data reduction using the BerSANS software and yielded one-dimensional scattering intensities I(q). The scattering curves of all samples were buffer subtracted using the software PRIMUS36 and the radii of gyration were extracted by the Guinier approximation. For SANS data sets at 100% D2O molecular mass estimates were obtained from the forward scattering (I0), with the contrast and partial specific volume as determined from the solution components and protein sequence using the MULCH server (http://smb-research.smb.usyd.edu.au/NCVWeb/)39. All SANS and SAXS scattering parameters have been included in Tables 1 and 2 according to the recommended publication guidelines for small-angle scattering studies40.Model calculation from SANS data (ACA8 in stealth carrier nanodiscs)The conformational flexibility of ACA8 in its activated state was probed using the ensemble optimized modeling (EOM) program20. A homology model of ACA8core (130aa-1074aa) was generated with the program Phyre241 using the聽SERCA structure in E2 conformation (pdb:3b9b). A pool of 10,000 ACA8 full-length models with the different conformations of the regulatory domain was generated with RANCH20 by using the ACA8core homology model and the crystal structure of the regulatory domain (pdb:4aqr) as input domains. Theoretical scattering intensity of each model was computed with CRYSON42. 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CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859鈥?865 (2008).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsWe thank Franz Hagn (Munich) for advice on nanodisc preparation, Poul Nissen and Thomas Boesen (Aarhus) for plasmids, Simon Newstead (Oxford) for yeast cells and Katharina Veith for technical assistance. The synchrotron SAXS data were collected at beamline P12 operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). The SANS data were collected at the SANS-1 instrument operated by HZG and FRM II at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany. The authors gratefully acknowledge the financial support provided by HZG to perform the neutron scattering measurements at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany. We are grateful to the staff at SANS-1 (MLZ, Munich) and P12 (EMBL, Hamburg). We acknowledge access to the Sample Preparation and Characterization (SPC) Facility of EMBL, Hamburg. VTF acknowledges the UK Engineering and Physical Sciences Research Council (EPSRC) for grants GR/R99393/01 and EP/C015452/1 that originally funded the Deuteration Laboratory within ILL鈥檚 Life Sciences Group that produced deuterated material for this study. The authors thank Professor William Dowhan and his group at the University of Texas, USA for their kind contribution in providing the original PC-producing E. coli strain, which was further mutated for production of deuterated PC in Grenoble. This research was funded by an Emmy Noether grant (to H.T.) and the excellence cluster 鈥楾he Hamburg Center for Ultrafast Imaging - Structure, Dynamics and Control of Matter at the Atomic Scale鈥?of the Deutsche Forschungsgemeinschaft (DFG EXC 1074). The Heinrich Pette Institute, Leibniz Institute for Experimental Virology is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health. J.H. and C.U. are funded by the Leibniz Association through SAW-2014-HPI-4 grant.Author informationAuthor notesThese authors contributed equally: Inokentijs Josts, Johannes Heidemann, Haydyn D. Mertens.AffiliationsThe Hamburg Centre for Ultrafast Imaging Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146, Hamburg, GermanyJulius Nitsche,聽Inokentijs Josts聽 聽Henning TidowHeinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistrasse 52, 20251, Hamburg, GermanyJohannes Heidemann聽 聽Charlotte UetrechtEuropean Molecular Biology Laboratory Hamburg, Notkestrasse 85, 22607, Hamburg, GermanyHaydyn D. Mertens聽 聽Dmitri I. SvergunBiofilms- Research Center for Biointerfaces, Department of Biomedical Science, Faculty of Health and Society, Malm枚 University, Malm枚, 20506, SwedenSelma MaricLife Sciences Group, Institut Laue鈥揕angevin, 6 Rue Jules Horowitz, 38042, Grenoble, FranceMartine Moulin,聽Michael Haertlein聽 聽V. Trevor ForsythGerman Engineering Materials Science Centre (GEMS) at Heinz Maier-Leibnitz Zentrum (MLZ), Helmholtz-Zentrum Geesthacht, Lichtenbergstr. 1, 85747, Garching bei M眉nchen, GermanySebastian BuschSchool of Life Sciences, Keele University, Staffordshire, ST5 5BG, EnglandV. Trevor ForsythEuropean XFEL GmbH, Holzkoppel 4, 22869, Schenefeld, GermanyCharlotte UetrechtAuthorsJulius NitscheView author publicationsYou can also search for this author in PubMed聽Google ScholarInokentijs JostsView author publicationsYou can also search for this author in PubMed聽Google ScholarJohannes HeidemannView author publicationsYou can also search for this author in PubMed聽Google ScholarHaydyn D. MertensView author publicationsYou can also search for this author in PubMed聽Google ScholarSelma MaricView author publicationsYou can also search for this author in PubMed聽Google ScholarMartine MoulinView author publicationsYou can also search for this author in PubMed聽Google ScholarMichael HaertleinView author publicationsYou can also search for this author in PubMed聽Google ScholarSebastian BuschView author publicationsYou can also search for this author in PubMed聽Google ScholarV. Trevor ForsythView author publicationsYou can also search for this author in PubMed聽Google ScholarDmitri I. SvergunView author publicationsYou can also search for this author in PubMed聽Google ScholarCharlotte UetrechtView author publicationsYou can also search for this author in PubMed聽Google ScholarHenning TidowView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsConceptualization, J.N. and H.T.; Methodology, J.N., I.J., S.M. and H.T.; Investigation, J.N., I.J., J.H., S.M., H.D.M., M.M., M.H., V.T.F., S.B. and H.T.; Writing鈥擮riginal Draft, J.N. and H.T.; Writing鈥擱eview Editing, J.N., I.J., H.D.M., D.I.S., S.B., V.T.F., C.U. and H.T.; Resources, D.I.S., C.U. and H.T.; Funding Acquisition, V.T.F. and H.T.; Supervision, H.T.Corresponding authorCorrespondence to Henning Tidow.Ethics declarations Competing interests The authors declare no competing interests. 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If material is not included in the article鈥檚 Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleNitsche, J., Josts, I., Heidemann, J. et al. Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin. Commun Biol 1, 206 (2018). https://doi.org/10.1038/s42003-018-0203-7Download citationReceived: 04 June 2018Accepted: 26 October 2018Published: 26 November 2018DOI: https://doi.org/10.1038/s42003-018-0203-7 Selma Maric, Tania Kjellerup Lind, Manfred Roman Raida, Eva Bengtsson, Gunilla Nordin Fredrikson, Sarah Rogers, Martine Moulin, Michael Haertlein, V. Trevor Forsyth, Markus R. Wenk, Thomas G眉nther Pomorski, Thomas Arnebrant, Reidar Lund Marit茅 C谩rdenas Scientific Reports (2019) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.