Skip to main content
Dryad logo

Data from: Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence

Citation

Mohammadi, Shabnam et al. (2022), Data from: Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence, Dryad, Dataset, https://doi.org/10.5061/dryad.sqv9s4n68

Abstract

A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitution’s effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases.

Methods

Construction of expression vectors.

ATP1A1 and ATP1B1 wild-type sequences for eight selected tetrapod species were synthesized by InvitrogenTM GeneArt. ATP1A1/B1 sequences used in these constructs can be found under the following accession numbers: Rattus norvegicus (ATP1A1 – X05882 ; ATP1B1 – NM013113.2), Chinchilla lanigera (ATP1A1 – XM005389040; ATP1B1 – XM005398203), Rhabdophis subminiatus (ATP1A1 – MT928191; ATP1B1 – ON168934), Xenodon rhabdocephalus (ATP1A1 – MT928200; ATP1B1 – ON168935), Varanus exanthematicus (ATP1A1 – MT928184; ATP1B1 – ON168936), Tupinambis teguixin (ATP1A1 – MT928189; ATP1B1 – ON168937), Struthio camelus (ATP1A1 – XM009675281; ATP1B1 – XM009675170), Pterocles gutturalis (ATP1A1 – XM010081314; ATP1B1 – XM010078905). The 𝛽1-subunit genes were inserted into pFastBac Dual expression vectors (Life Technologies) at the p10 promoter with XhoI and PaeI (FastDigest Thermo ScientificTM) and then control sequenced. The α1-subunit genes were inserted at the PH promoter of vectors already containing the corresponding 𝛽1-subunit proteins using In-Fusion® HD Cloning Kit (Takara Bio, USA Inc.) and control sequenced. All resulting vectors had the α1-subunit gene under the control of the PH promoter and a 𝛽1-subunit gene under the p10 promoter. The resulting eight vectors were then subjected to site-directed mutagenesis (QuickChange II XL Kit; Agilent Technologies, La Jolla, CA, USA) to introduce the codons of interest. In total, 21 vectors were produced.

Generation of recombinant viruses and transfection into Sf9 cells.

Escherichia coli DH10bac cells harboring the baculovirus genome (bacmid) and a transposition helper vector (Life Technologies) were transformed according to the manufacturer’s protocol with expression vectors containing the different gene constructs. Recombinant bacmids were selected through PCR screening, grown, and isolated. Subsequently, Sf9 cells (4 x 105 cells*ml) in 2 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) were transfected with recombinant bacmids using Cellfectin reagent (Life Technologies). After a three-day incubation period, recombinant baculoviruses were isolated (P1) and used to infect fresh Sf9 cells (1.2 x 106 cells*ml) in 10 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) with 15 mg/ml gentamycin (Roth, Karlsruhe, Germany) at a multiplicity of infection of 0.1. Five days after infection, the amplified viruses were harvested (P2 stock).

Preparation of Sf9 membranes.

For production of recombinant NKA, Sf9 cells were infected with the P2 viral stock at a multiplicity of infection of 103. The cells (1.6 x 106 cells*ml) were grown in 50 ml of Insect-Xpress medium (Lonza, Walkersville, MD, USA) with 15 mg/ml gentamycin (Roth, Karlsruhe, Germany) at 27°C in 500 ml flasks (35). After 3 days, Sf9 cells were harvested by centrifugation at 20,000 x g for 10 min. The cells were stored at -80 °C and then resuspended at 0 °C in 15 ml of homogenization buffer (0.25 M sucrose, 2 mM EDTA, and 25 mM HEPES/Tris; pH 7.0). The resuspended cells were sonicated at 60 W (Bandelin Electronic Company, Berlin, Germany) for three 45 s intervals at 0 °C. The cell suspension was then subjected to centrifugation for 30 min at 10,000 x g (J2-21 centrifuge, Beckmann-Coulter, Krefeld, Germany). The supernatant was collected and further centrifuged for 60 m at 100,000 x g at 4 °C (Ultra- Centrifuge L-80, Beckmann-Coulter) to pellet the cell membranes. The pelleted membranes were washed once and resuspended in ROTIPURAN® p.a., ACS water (Roth) and stored at -20 °C. Protein concentrations were determined by Bradford assays using bovine serum albumin as a standard. Three biological replicates were produced for each NKA construct.

Verification by SDS-PAGE/western blotting.

For each biological replicate, 10 mg of protein were solubilized in 4x SDS-polyacrylamide gel electrophoresis sample buffer and separated on SDS gels containing 10% acrylamide. Subsequently, they were blotted on nitrocellulose membrane (HP42.1, Roth). To block non-specific binding sites after blotting, the membrane was incubated with 5% dried milk in TBS-Tween 20 for 1 h. After blocking, the membranes were incubated overnight at 4 °C with the primary monoclonal antibody α5 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA). Since only membrane proteins were isolated from transfected cells, detection of the α subunit also indicates the presence of the β subunit. The primary antibody was detected using a goat-anti-mouse secondary antibody conjugated with horseradish peroxidase (Dianova, Hamburg, Germany). The staining of the precipitated polypeptide-antibody complexes was performed by addition of 60 mg 4-chloro-1 naphtol (Sigma-Aldrich, Taufkirchen, Germany) in 20 ml ice-cold methanol to 100 ml phosphate buffered saline (PBS) containing 60 ml 30% H2O2

Ouabain inhibition assay.

To determine the sensitivity of each NKA construct against cardiotonic steroids (CTS), we used the water-soluble cardiac glycoside, ouabain (Acrōs Organics), as our representative CTS. 100 ug of each protein was pipetted into each well in a nine-well row on a 96-well microplate (Fisherbrand) containing stabilizing buffers (see buffer formulas in Petschenka et al. 2013). Each well in the nine-well row was exposed to exponentially decreasing concentrations of ouabain (10-3 M, 10-4 M, 10-5 M, 10-6 M, 10-7 M, 10-8 M, dissolved in distilled H2O), plus distilled water only (experimental control), and a combination of an inhibition buffer lacking KCl and 10-2 M ouabain to measure background protein activity [48]. The proteins were incubated at 37°C and 200 rpms for 10 minutes on a microplate shaker (Quantifoil Instruments, Jena, Germany). Next, ATP (Sigma Aldrich) was added to each well and the proteins were incubated again at 37°C and 200 rpms for 20 minutes. The activity of NKA following ouabain exposure was determined by quantification of inorganic phosphate (Pi) released from enzymatically hydrolyzed ATP. Reaction Pi levels were measured according to the procedure described in Taussky and Shorr (1950) (see Petschenka et al. 2013). All assays were run in duplicate and the average of the two technical replicates was used for subsequent statistical analyses. Absorbance for each well was measured at 650 nm with a plate absorbance reader (BioRad Model 680 spectrophotometer and software package). 

ATP hydrolysis assay.

To determine the functional efficiency of different NKA constructs, we calculated the amount of Pi hydrolyzed from ATP per mg of protein per minute. The measurements (the mean of two technical replicates) were obtained from the same assay as described above. In brief, absorbance from the experimental control reactions, in which 100 mg of protein was incubated without any inhibiting factors (i.e., ouabain or buffer excluding KCl), were measured and translated to mM Pi from a standard curve that was run in parallel (1.2 mM Pi, 1 mM Pi, 0.8 mM Pi, 0.6 mM Pi, 0.4 mM Pi, 0.2 mM Pi, 0 mM Pi). 

Statistical analyses of functional data.  

ATPase activity in the presence and absence of the CTS ouabain was measured following Petschenka et al. (2013). Background phosphate absorbance levels from reactions with inhibiting factors were used to calibrate phosphate absorbance. For ouabain sensitivity measurements, these calibrated absorbance values were converted to percentage non-inhibited NKA activity based on measurements from the control wells (as above). For each of the 3 biological replicates, log10 IC50 values were estimated using a four-parameter logistic curve, with the top asymptote set to 100 and the bottom asymptote set to zero, using the nlsLM function of the minipack.lm library in R.  To measure baseline recombinant protein activity, the calculated Pi concentrations of 100 mg of protein assayed in the absence of ouabain were converted to nmol Pi/mg protein/min.

1. Petschenka G, Fandrich S, Sander N, Wagschal V, Boppré M, Dobler S. Stepwise evolution of resistance to toxic cardenolides via genetic substitutions in the Na+/K+‐ATPase of milkweed butterflies (Lepidoptera: Danaini). Evolution. 2013;67: 2753–2761.

2. Taussky HH, Shorr E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953;202: 675–685.

Funding

National Institutes of Health, Award: R01-GM115523

National Institutes of Health, Award: R01-HL087216

National Institutes of Health, Award: F32– 695 HL149172

National Science Foundation, Award: OIA-1736249

Deutsche Forschungsgemeinschaft, Award: Do 517/10-1

Alexander von Humboldt-Stiftung, Award: Mohammadi 2018-2019