Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin

The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3ecto), a signalling-competent Furin1–Furin2 (Fu1–Fu2) fragment of Rspo2 (Rspo2Fu1–Fu2), and Rspo2Fu1–Fu2 in complex with ZNRF3ecto, or RNF43ecto. A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3ecto and RNF43ecto surface. Rspo binding enhances dimerization of ZNRF3ecto but not of RNF43ecto. Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2

The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling

Substrate specificities of AtAPY1 and AtAPY1-δTM.

<p>Activities (1 U = 1 μmol P_i /min) of AtAPY1 (A) and AtAPY1-δTM (B) were determined in the presence of various substrates (3 mM each) using the discontinuous apyrase activity assay. The means <u>+</u> SD of duplicates from one assay are shown. Different letters above the columns indicate mean values that are significantly different from one other (one-way ANOVA and Tukey test; p < 0.05). Each data set is representative of three independent activity assays.</p

Substrate specificity of AtAPY1-δTM in the presence of CaM.

<p>Activities (1 U = 1 μmol P_i /min) of AtAPY1-δTM were determined in the presence of various substrates (3 mM each) and 1 μM CaM using the discontinuous apyrase activity assay. The means <u>+</u> SD of three independent assays are shown. Different letters above the columns indicate mean values that are significantly different from one other (one-way ANOVA and Tukey test; p < 0.05).</p

Putative three-dimensional structural model of the AtAPY1 ecto-domain.

<p>The structural model was colored using the ConSurf server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#pone.0115832.ref083" target="_blank">83</a>] and 300 random NTPDase sequences sharing between 20 and 80% sequence identity. The N-terminal (NT) and C-terminal (CT) residue are marked. Three conserved cysteine bridges are predicted for the C-terminal lobe, while the non-conserved C87 is likely to be free. A single N-acetyl glucosamine group is drawn to indicate the solvent-exposed position of the single potential N-glycosylation site N333 in the back. Next to the active site Mg²⁺ ion (gray sphere) a vanadate ion is shown in the active site to highlight its likely competitive inhibition mechanism. The putative CaM-binding site (CBS) ¹⁶⁹VRELLKGRSRLK¹⁸⁰ is depicted as red sticks. An approximate binding mode for GDP is shown in half transparency. F366 and N415 are strong candidates for base sandwich binding. Y418 might be involved in base binding as well but would have to adopt a different side chain conformation than that assigned by the MODELLER program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#pone.0115832.ref048" target="_blank">48</a>].</p

Protein sequence characteristics of AtAPY1.

<p>The complete AtAPY1 protein sequence is shown. The transmembrane region (TM) is underlined and the putative N-glycosylation site is marked in green. The ACRs are boxed. Six of the seven cysteines (black asterisks) are highly conserved and highlighted in yellow. The putative calmodulin-binding site is circled in orange. A black triangle marks where the sequence of AtAPY1-δTM begins.</p

Detection of extracted AtAPY1-GFP.

<p>A Western blot analysis of two different crude protein extracts before (= in) and after (= out) the immobilization by GFP-multiTrap is shown. Equal volumes of extract (15 μL each) were loaded per lane, its proteins subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane and incubated with antibodies to GFP. The arrows mark the signals of the AtAPY1-GFP fusion protein and free GFP, respectively. The explanation of the colors in the schematic representation of AtAPY1-GFP can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115832#pone.0115832.g002" target="_blank">Fig. 2</a>. Recombinant GFP (19 ng) served as a positive control and quantitative reference for densitometric evaluation of signal intensities. With this, the total amounts of bound AtAPY1-GFP from 100 μL extract 1 and 2 were calculated as 130 ng and 22 ng, respectively. The image shows bands from the same exposure of the same membrane, but non-pertinent lanes were cropped as indicated by vertical lines. The shown signals are representative of at least five separate Western blot analyses of different GFP-multiTrap immobilization experiments.</p

K_m values.

<p>The mean K_m values are listed ± SD. The K_m values for GDP, UDP and IDP were all significantly different from each other (p < 0.0001; one-way ANOVA test and Tukey test).</p><p>¹The mean of the K_m value was calculated from six separate experiments. The means were not statistically different from each other (p < 0.001; one-way ANOVA). AtAPY1-GFP purified from three different protein extracts (biological repeats) was analyzed. One, two and three separate experiments were run with each protein extract, respectively.</p><p>²The mean of the K_m value was calculated from six separate experiments. The means were not statistically different from each other (p < 0.001; one-way ANOVA). AtAPY1-GFP purified from two different protein extracts (biological repeat) was analyzed. Two and four separate experiments were run with each protein extract, respectively.</p><p>³The mean of the K_m value was calculated from three separate experiments. The means were not statistically different from each other (p < 0.01; one-way ANOVA). AtAPY1-GFP purified from two different protein extracts (biological repeat) was analyzed resulting in one technical and one biological repeat.</p><p>K_m values.</p

Influence of divalent metal ions on AtAPY1 activity.

<p>Enzyme activities were determined in the presence of 3 mM UDP using the discontinuous apyrase activity assay. The activity of AtAPY1 (1 U = 1 μmol P_i /min) was measured in the absence or presence of either 1 mM CaCl₂, CuCl₂, MgCl₂, MnCl₂, NiCl₂ or ZnCl₂. The control (-) shows the activity without the addition of any divalent ions. The means <u>+</u> SD of duplicates from one assay are shown. Different letters above the columns indicate mean values that are significantly different from one other (one-way ANOVA and Tukey test; p < 0.01). Data are representative of two activity assays.</p