DOI: 10.5061/dryad.sqv9s4n68
title: Data from: Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence

description: 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.
description: <strong>Construction of
 expression vectors.</strong>
 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: <em>Rattus
 norvegicus</em> (ATP1A1 – X05882 ; ATP1B1 – NM013113.2),
 <em>Chinchilla lanigera </em>(ATP1A1 – XM005389040; ATP1B1 –
 XM005398203), <em>Rhabdophis subminiatus</em> (ATP1A1 –
 MT928191; ATP1B1 – ON168934), <em>Xenodon rhabdocephalus</em>
 (ATP1A1 – MT928200; ATP1B1 – ON168935), <em>Varanus exanthematicus</em>
 (ATP1A1 – MT928184; ATP1B1 – ON168936), <em>Tupinambis teguixin</em>
 (ATP1A1 – MT928189; ATP1B1 – ON168937), <em>Struthio camelus</em> (ATP1A1
 – XM009675281; ATP1B1 – XM009675170), <em>Pterocles
 gutturalis</em> (ATP1A1 – XM010081314; ATP1B1 – XM010078905).
 The ?1-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 ?1-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
 ?1-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. <strong>Generation of recombinant viruses and
 transfection into Sf9 cells.</strong>
 <em>Escherichia
 coli</em><em>
 </em>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
 10<sup>5</sup> 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 10<sup>6</sup> 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). <strong>Preparation of Sf9
 membranes.</strong> For production of recombinant NKA, Sf9 cells
 were infected with the P2 viral stock at a multiplicity of infection of
 10<sup>3</sup>. The cells (1.6 x 10<sup>6</sup>
 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.
 <strong>Verification by
 SDS-PAGE/western blotting.</strong>
 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%
 H<sub>2</sub>O<sub>2</sub>.  <strong>Ouabain inhibition assay.</strong> 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<sup>-3</sup> M, 10<sup>-4</sup> M, 10<sup>-5</sup> M, 10<sup>-6</sup> M, 10<sup>-7</sup> M, 10<sup>-8</sup> M, dissolved in distilled H<sub>2</sub>O), plus distilled water only (experimental control), and a combination of an inhibition buffer lacking KCl and 10<sup>-2</sup> 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).  <strong>ATP hydrolysis assay.</strong> 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).  <strong>Statistical analyses of functional data.  </strong> 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 IC<sub>50</sub> 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.
description: # Data from: Constraints on the evolution of toxin-resistant Na,K-ATPases
 have limited dependence on sequence divergence
 [https://doi.org/10.5061/dryad.sqv9s4n68](https://doi.org/10.5061/dryad.sqv9s4n68) ## Description of the data and file structure This Corrected TEG+Q111T.xlsx file was generated on 2023-04-21 by Shabnam Mohammadi GENERAL INFORMATION 1\. Title of Dataset: Corrected TEG+Q111T 2\. Author Information A. Principal Investigator Contact Information Name: Peter Andolfatto Institution: Columbia University Email: [pa2543@columbia.edu](mailto:pa2543@columbia.edu)  B. Associate or Co-investigator Contact Information Name: Shabnam Mohammadi Institution: Universität Hamburg Email: [shab.mohammadi@gmail.com](mailto:shab.mohammadi@gmail.com) 3\. Date of data collection: between 2022-12-01 and 2023-04-01  4\. Geographic location of data collection: Universität Hamburg  5\. Information about funding sources that supported the collection of the data: National Institutes of Health to PA (R01-GM115523), JFS (R01-HL087216), and SM (F32– 695 HL149172), the National Science Foundation (OIA-1736249) to JFS; the Deutsche Forschungsgemeinschaft (Do 517/10-1) to SD; the Alexander von Humboldt Foundation to SM. SHARING/ACCESS INFORMATION Links to publications that cite or use the data: doi:10.1101/2021.11.29.470343 Recommended citation for this dataset:  Mohammadi S, Yang L, Herrera-Álvarez S, del Pilar Rodríguez-Ordoñez M, Zhang K, Storz JF, Dobler S, Crawford AJ, Andolfatto P. 2021. Epistasis is not a strong constraint on the recurrent evolution of toxin-resistant Na+, K+-ATPases among tetrapods. bioRxiv. doi:10.1101/2021.11.29.470343 DATA & FILE OVERVIEW 1\. File List: Corrected TEG+Q111T.xlsx 2\. Relationship between files, if important: This file contains the corrected data for one of the protein constructs in the file NKA_Enzyme_Assays_Raw_Data.xlsx 3\. Additional related data collected that was not included in the current data package: N/A 4\. Are there multiple versions of the dataset? No METHODOLOGICAL INFORMATION 1\. Description of methods used for collection/generation of data:  [https://doi.org/10.1101/2021.11.29.470343](https://doi.org/10.1101/2021.11.29.470343) 2\. Methods for processing the data:  Background phosphate absorbance levels from reactions with inhibiting factors were used to calibrate phosphate absorbance in wells measuring ouabain inhibition and in the control wells. For ouabain sensitivity measurements, calibrated absorbance values were converted to percentage non-inhibited Na+,K+-ATPases activity based on measurements from the control wells. These data were plotted and log IC50 values were obtained for each biological replicate from nonlinear fitting using a four-parameter logistic curve, with the top asymptote set to 100 and the bottom asymptote set to zero. Curve fitting was performed with the nlsLM function of the minipack.lm library in R. For comparisons of recombinant protein ATPase activity, the calculated Pi concentrations of 100 ug of protein assayed in the absence of ouabain were converted to nmol Pi/mg protein/min.  3\. Instrument- or software-specific information needed to interpret the data:  nlsLM function of the minipack.lm library in R 4\. Standards and calibration information, if appropriate: N/A 5\. Environmental/experimental conditions: N/A 6\. Describe any quality-assurance procedures performed on the data: Full vector sequence for this construct was validated by Addgene (plasmid # 187435). 7\. People involved with sample collection, processing, analysis and/or submission: Shabnam Mohammadi DATA-SPECIFIC INFORMATION FOR: Corrected TEG+Q111T.xlsx 1\. Number of variables: Data were collected for 3 biological replicates (A-B) of 1 recombinant protein (Tegu+Q111T). Data for each biological replicate was collected in duplicate (2 technical replicates).  2\. Number of cases/rows: N/A 3\. Variable List: The Excel spreadsheet contains raw plate reader data and downstream calculations that led to IC50 values (described above) and ATPase activity values. For the ATPase activity values, control and background inorganic phosphate (Pi) was interpolated in from standard curve. The Pi standard curve is included on this sheet, to the right of the raw plate data.  4\. Missing data codes: N/A 5\. Specialized formats or other abbreviations used: N/A File: Corrected_TEG_Q111T.xlsx: contains the corrected data for one of the protein constructs in the original data file "NKA_Enzyme_Assays_Raw_Data.xlsx" File: NKA_Enzyme_Assays_Raw_Data.xlsx Number of variables: Data were collected for 3 biological replicates (A-B) of 21 recombinant proteins (Rat; Chinchilla; Xenodon; Rhabdophis; Varanus; Tupinambis; Ostrich; Sandgrouse; Rat+R111E; Rat+D122H; Chinchilla+N122D; Xenodon+H122D; Xenodon+H122T; Xenodon+H111E; Rhabdophis+E111H; Varanus+T111H; Varanus+N122H; Tupinambis+Q111T; Tupinambis+H122D; Ostrich+Q111R; Sandgrouse+R111Q). An additional set of data were obtained from doi:10.1101/2020.08.04.234435 for 6 biological replicates (A-F) of 3 recombinant proteins (S; S+Q111R; S+N122D). Data for each biological replicate was collected in duplicate (2 technical replicates). The first sheet of the Excel spreadsheet contains raw plate reader data and downstream calculations that led to IC50 values (described above). Three recombinant proteins were catalytically inactive and had no measurable IC50 values (Varanus+N122H; Tupinambis+Q111T; Tupinambis+H122D). The second sheet contains a subset of these data that were calculated to obtain ATPase activity values. Control and background inorganic phosphate (Pi) was interpolated in from standard curve in GraphPad Prism 9 software. Pi standard curves for each plate are included on this sheet, to the right of the activity data. One recombinant protein (Tupinambis+Q111T) exhibited low expression by western blot.