Saposin-Lipoprotein Scaffolds for Structure Determination of Membrane Transporters.

Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin-lipid-protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al., 2016). The method is fast and requires few resources. The saposin-lipid-scaffold adapts to various sizes of transmembrane regions during self-assembly, forming a minimal lipid nanoparticle. This results in the formation of a well-defined membrane protein-lipid complex, which is desirable for structural characterization. Here, we describe a protocol to reconstitute the sarco-endoplasmic reticulum calcium ATPase (SERCA) into Salipro nanoparticles. The complex formation is analyzed using negative stain electron microscopy (EM), allowing to quickly determine an initial structure of the membrane protein and to evaluate sample conditions for structural studies using single-particle cryo-EM in a detergent-free environment.

Preparation of Ca(2+)-ATPase1a Enzyme from Rabbit Sarcoplasmic Reticulum.

The SERCA calcium ATPase, probably the most well-investigated membrane protein both from a biophysical and structural view, can be purified from native source in substantial quantities, making it a favorable target when conducting biochemical experiments and structure determination, e.g., by X-ray crystallography.

Calcium Uptake in Crude Tissue Preparation.

The various isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) are responsible for the Ca(2+) uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca(2+) as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated (45)Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells.

Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.

Optimisation of recombinant production of active human cardiac SERCA2a ATPase.

Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca(2+) translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins.

Identification of calcium-transporting ATPases of Entamoeba histolytica and cellular localization of the putative SERCA.

Calcium has an important role on signaling of different cellular processes in the protozoa parasite Entamoeba histolytica, including development and pathogenesis. However, the systems that control calcium responses in this parasite are incompletely understood. Calcium-ATPases (Ca(2+)-ATPases) are proteins that play an important role in calcium homeostasis by catalyzing the active efflux of this ion from cytoplasm and are essential to the correct functioning of the cell machinery. Here, we reported the identification of five E. histolytica genes encoding putative Ca(2+)-ATPases, three related to PMCA, and two related to organellar ATPases. RT-PCR assays showed that all those genes are expressed in trophozoites and specific antibodies against the SERCA-like member located this protein in a continuous cytoplasmic network, supporting the hypothesis that it corresponds to the Ca(2+)-ATPase responsible to sequester calcium in the endoplasmic reticulum of this parasite.

Avidin functionalized maghemite nanoparticles and their application for recombinant human biotinyl-SERCA purification.

We report on the surface characterization, functionalization, and application of stable water suspensions of novel surface active maghemite nanoparticles (SAMNs), characterized by a diameter of 11 ± 2 nm and possessing peculiar colloidal properties and surface interactions. These features permitted the acquisition of titration curves and aqueous UV-vis spectra and suggested a role played by surface under-coordinated iron atoms. This new class of nanoparticles was obtained through an easy, inexpensive, one-step, green procedure and functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution. Bound avidin was determined by measuring the disappearance of free avidin absorbance at 280 nm, as a function of increasing nanoparticle concentration, showing the presence of 10 ± 3 avidin molecules per nanoparticle. The biological activity of the SAMN@avidin complex was evaluated and the number of available biotin binding sites was determined, using biotinyl-fluorescein as a probe, showing that each bound avidin molecule is able to bind 2.8 ± 0.8 biotin molecules, confirming the maintenance of biological activity and excellent binding capacity of the SAMN@avidin complex. Furthermore a Langmuir isotherm model was used to describe the biomolecule specific monolayer adsorption onto the particle surface, and in the case of avidin, the maximum adsorption capacity was 100 ± 27 µg avidin/mg SAMN, whereas the binding constant is 45.18 µL µg(-1). The SAMN@avidin complex was characterized by UV-vis spectroscopy, quartz crystal microbalance, FTIR spectroscopy, and transmission electron microscopy. Finally, SAMN@avidin was applied for the large scale purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca(2+)-ATPase (hSERCA-2a), expressed by Saccharomyces cerevisiae. The protein was magnetically purified, and about 500 µg of a 70% pure hSERCA-2a were recovered from 4 L of yeast culture, with a purification yield of 64%.

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases.

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Heterologous expression and affinity purification of eukaryotic membrane proteins in view of functional and structural studies: The example of the sarcoplasmic reticulum Ca(2+)-ATPase.

Heterologous SERCA1a Ca(2+)-ATPase (sarco-endoplasmic reticulum Ca(2+)-adenosine triphosphatase isoform 1a) from rabbit was expressed in yeast Saccharomyces cerevisiae as a fusion protein, with a biotin acceptor domain (BAD) linked to the SERCA C-terminus by a thrombin cleavage site. Thanks to the pYeDP60 vector, the recombinant protein was expressed under the control of a galactose-inducible promoter. Biotinylation of the protein occurred directly in yeast. Optimizing the number of galactose induction steps and increasing the amount of Gal4p transcription factor both improved expression. Lowering the temperature from 28 to 18 degrees C during expression enhanced the recovery of detergent-extractible active protein. In the "light membrane fraction," thought to mainly contain internal membranes, we are able to recover about 14-18 mg Ca(2+)-ATPase per liter of yeast culture in a bioreactor. Solubilization of this membrane fraction by n-dodecyl beta-D: -maltopyranoside (DDM) allowed us to recover the largest amount of active protein. The in vivo biotinylated recombinant protein was then bound to a streptavidin-Sepharose resin. Selective elution of the biotinylated SERCA1a was carried out after thrombin action on the resin-bound protein. We were able to obtain 200-500 microg/L of yeast culture of a 50% pure SERCA1a that displays an ATPase activity similar to that of the native rabbit Ca(2+)-ATPase. To succeed in crystallization, an additional size exclusion chromatography step was necessary. This step increases purity to 70%, removes aggregated protein and exchanges DDM for C(12)E(8).

Use of glycerol-containing media to study the intrinsic fluorescence properties of detergent-solubilized native or expressed SERCA1a.

Rapid irreversible inactivation of Ca (2+)-free states of detergent-solubilized SERCA1a (sarco-endoplasmic reticulum calcium ATPase 1a) has so far prevented the use of Trp fluorescence for functional characterization of this ATPase after its solubilization in various detergents. Here we show that using 20-40% glycerol for protection makes this fluorescence characterization possible. Most of the ligand-induced Trp fluorescence changes previously demonstrated to occur for SERCA1a embedded in native sarcoplasmic reticulum membranes were observed in the combined presence of glycerol and detergent, although the results greatly depended on the detergent used, namely, octaethylene glycol mono- n-dodecyl ether (C 12E 8) or dodecyl maltoside (DDM). In particular, at pH 6, we found a C 12E 8-dependent unexpectedly huge reduction in SERCA1a affinity for Ca (2+). We suggest that a major reason for the different effects of the two detergents is that high concentrations of C 12E 8, but not of DDM, slow down the E2 to E1 transition in solubilized and delipidated SERCA1a. Independently of the characterization of the specific effects of various detergents on SR vesicles, our results open the way to functional characterization by Trp fluorescence of heterologously expressed and purified mutants of SERCA1a in the presence of detergent, without their preliminary reconstitution into liposomes. As an example, we used the E309Q mutant to demonstrate our previous suspicion that Ca (2+) binding to Site I of SERCA1a in fact slightly reduces Trp fluorescence, and consequently that the rise in this fluorescence generally observed when two Ca (2+) ions bind to WT SERCA1a mainly reflects Ca (2+) binding at Site II of SERCA1a.

Crystal structure of D351A and P312A mutant forms of the mammalian sarcoplasmic reticulum Ca(2+) -ATPase reveals key events in phosphorylation and Ca(2+) release.

In recent years crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and AlF(4)(-) bound (3.5-A resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-A resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca(2+) was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca(2+)-translocating Ca(2)E1P-->E2P transition, which seems to be due to a stabilization of Ca(2)E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro(312) with alanine in P312A.

Oligomerisation of sarcoplasmic reticulum Ca2+-ATPase monomers from skeletal muscle.

The fast-twitch SERCA1 isoform of the sarcoplasmic reticulum Ca(2+)-ATPase was purified to homogeneity and conjugated to peroxidase. The SERCA1 probe showed high affinity binding to the immobilized monomeric enzyme, but not crosslinker-stabilized oligomers. This suggests a preferential complex formation via homo-dimerization, rather than interactions with established oligomeric structures.

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain.

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.