Progesterone binding to the alpha1-subunit of the Na/K-ATPase on the cell surface: insights from computational modeling

Abstract

Progesterone triggers the resumption of meiosis in the amphibian oocyte through a signaling system at the plasma membrane. Analysis of [(3)H]ouabain and [(3)H]progesterone binding to the plasma membrane of the Rana pipiens oocyte indicates that progesterone competes with ouabain for a low affinity ouabain binding site on a 112kDa alpha1-subunit of the membrane Na/K-ATPase. Published amino acid sequences from both low and high affinity ouabain binding alpha1-subunits are compared, together with published site-directed mutagenesis studies of ouabain binding. We propose that the progesterone binding site is located in the external loop (23 amino acids) between the M1-M2 transmembrane helices. Analysis of loop topology and the countercurrent hydrophobicity/polarity gradients within the M1-M2 loop further suggest that the polar beta and hydrophobic alpha surfaces of the planar progesterone molecule interact with opposite sides of the amino acid loop. The 19-angular methyl group of progesterone is essential for activity; it could bind to the C-terminal region of the M1-M2 loop. Maximum biological activity requires formation of hydrogen-bond networks between the 3-keto group of progesterone and Arg(118), Asp(129) and possibly Glu(122-124) in the C-terminal region of the loop. The 20-keto group hydrogen may in turn hydrogen bond to Cys(111) near the M1 helix. Peptide flexibility undergoes a maximal transition near the midway point in the M1-M2 loop, suggesting that folding occurs within the loop, which further stabilizes progesterone binding.

Figure 1

Structures of Δ⁴-pregnane-3,20-dione (progesterone) and the cardiotonic steroid ouabain. Compounds with an unsaturated five-membered lactone ring are cardenolides and those with an unsaturated six-membered lactone ring are bufadienolides. In the formulae, bonds or atoms or groups above the plane of the ring system are depicted as solid wedges; those below the plane are depicted as open, dashed wedges.

Figure 2

The primary amino acid sequence of the complete (A) and truncated (B) graphics of the α-1 subunit of Xenopus laevis Na/K-ATPase in Scalable Vector graphics (SVG) format [17]. The sequence (primary accession # Q92127) was obtained from the nucleotide sequence of Xenopus laevis kidney epithelium [43] and neural plate cells [44]. The N and C termini of the α-subunit of the Na/K-ATPase are located intracellularly and the protein has 10 transmembrane domains and 2 large intracellular loops (see text). The intracellular N-terminal and C-terminal ends contain a white spacer to shorten the terminal sequence due to space limitations.

Figure 3

A comparison of 3D projection of the 23 amino acid sequence in the first external loop of the α1 subunit of the Na/K-ATPase of the high affinity ouabain binding isoform (human, upper cartoon) and low affinity ouabain binding isoform (Rat, lower cartoon). Plots were generated using Chem 3D Ultra v. 10.0 (Cambridgesoft.com). Colors indicate individual amino acids.

Figure 4

An expanded amino acid Scalable Vector Graphics (SVG) plot of the M1-M2 domain with the extracellular loop that joins the two transmembrane domains for the α1-isoform from X. laevis (Primary accession #Q92127). Arrows indicate residues identified as determinants of ouabain sensitivity for the sheep enzyme [13]; Cys¹¹³, Tyr¹¹⁷, Gln¹²¹, Pro¹²⁷, Asp¹³⁰, and Asn¹³¹. Residue numbers are based on the sequence for X. laevis [43,44]. The N-terminal intracellular domain (80 residues, primary accession # P04074)of the sheep α1 subunit is about 9 residues shorter than that of the Xenopus α1 subunit (89 residues).

Figure 5

The hydrophobicity (upper panel) and polarity (lower panel) gradients within the first external loop of the gradient for the α1 isomer illustrated in Figure 5. The sequence (residues 110-132, abscissa) was analyzed using ProtScale (expasy.org) and is shown as a function of the hydrophobicity or polariety score on the ordinate. The window was set at 5 and the algorithm of Kyte and Doolittle [18] was used to estimate hydrophobicity whereas the algorithm of Grantham [19], was used to measure polarity.

Figure 6

Protein sequence analysis of the first external loop (M1-M2) using hydrophobic cluster analysis [20]. The upper projection illustrates the sequence I¹⁰⁹ …Y¹³¹ (rat Primary accession # P06685) written on a classical α-helix (3.6 amino acids per turn). The lower projection indicates the hydrophobic cluster analysis after unrolling the helix (see text) with the hydrophobic clusters circled.

Figure 7

The average flexibility index along the first external loop is illustrated as a function of amino acid position in Xenopus laevis α1-subunit of Na/K-ATPase. The average flexibility index for the sequence (residues 110-132, abscissa) was calculated by the algorithm of Bhaskaran and Ponnuswamy [48] using ProtScale (expasy.org).

Figure 8

An illustration of the possible progesterone binding site within the first external loop of the low affinity ouabain binding isoform of the α1-subunit of the Na/K-ATPase. The graphic represents a view looking down on the cell surface, with an end view of the first (M1) and second (M2) transmembrane helices and the interconnecting loop of 23 amino acids. The peptide bonds between the helices and the external loop amino acids are discontinuous to allow for 90 degree rotation of the loop sequence. The arrows indicate the direction of insertion of the planar steroid into the loop. The amino acids in the external loop are color coded and the amino acids thought to be critical for ouabain binding are indicated in single letter codes (C¹¹¹, R¹¹⁸, S¹²², P¹²⁸, D¹²⁹, D¹³¹). The planar progesterone (shown on its side with the α hydrophobic face upward and to the left) interacts with hydrophobic clusters in the N-terminal region. The 19-methyl group (facing down and to the right) interacts with residues at the beginning of the transmembrane helix (M2). As proposed, a hydrogen-bonding network forms between the 3-keto group and the conserved residues Gln¹²⁰, Asn¹³¹ and Tyr¹¹⁸ in the first external loop of the α-1 subunit of the rat Na/KATPase.