Conservation of Cdc14 phosphatase specificity in plant fungal pathogens: implications for antifungal development

Abstract

Cdc14 protein phosphatases play an important role in plant infection by several fungal pathogens. This and other properties of Cdc14 enzymes make them an intriguing target for development of new antifungal crop treatments. Active site architecture and substrate specificity of Cdc14 from the model fungus Saccharomyces cerevisiae (ScCdc14) are well-defined and unique among characterized phosphatases. Cdc14 appears absent from some model plants. However, the extent of conservation of Cdc14 sequence, structure, and specificity in fungal plant pathogens is unknown. We addressed this by performing a comprehensive phylogenetic analysis of the Cdc14 family and comparing the conservation of active site structure and specificity among a sampling of plant pathogen Cdc14 homologs. We show that Cdc14 was lost in the common ancestor of angiosperm plants but is ubiquitous in ascomycete and basidiomycete fungi. The unique substrate specificity of ScCdc14 was invariant in homologs from eight diverse species of dikarya, suggesting it is conserved across the lineage. A synthetic substrate mimetic inhibited diverse fungal Cdc14 homologs with similar low µM K_i values, but had little effect on related phosphatases. Our results justify future exploration of Cdc14 as a broad spectrum antifungal target for plant protection.

Figure 1

Phylogenetic analysis of Cdc14. (a) Top, ribbon structure of the ScCdc14 catalytic domain (PDB 5XW5) with bound peptide substrate in ball and stick representation (red box). The N-terminal DSPn domain is colored magenta and the C-terminal DSPc domain containing the catalytic motif orange. The boxed region is enlarged in surface representation below showing the substrate binding site with key substrate recognition features labeled. (b) Taxonomic distribution of CDC14 gene family across Metazoa, Fungi, and Plantae. The height of the triangles indicates the relative representation of each lineage in the database, and the color of the triangles indicates the percentage of each lineage found to contain at least one copy of CDC14. The numbers to the right of each lineage name are the number of species containing one or more CDC14 gene/species in the database. The lineages in which no CDC14 copies were identified are bolded. (c) Unrooted maximum likelihood phylogenetic tree of Cdc14 genes.

Figure 2

The Cdc14 active site is highly conserved in plant pathogenic fungi. (a) Fungal lineages of the eight plant pathogen species chosen for study. (b) Structural model of ScCdc14 (PDB 5XW5) colored to map sequence conservation among the eight plant pathogen homologs from (a). Dark blue indicates identity, and light blue similarity, in all species. The yellow ball and stick phosphopeptide substrate molecule marks the active site and substrate binding groove location. (c) ScCdc14 amino acids within 4.5 Å of the substrate pSer, + 1 Pro, and + 3 Lys in structure 5XW5. Dashed lines between atoms indicate hydrogen bonds. (d) Clustal Omega alignment of the plant pathogen Cdc14 homologs from (a) with ScCdc14. Highlighted residues include those shown in (c), and others previously implicated in substrate recognition and specificity^,–. Colored symbols above highlighted residues indicate substrate feature(s) that each is known or predicted (+) to interact with.

Figure 3

A novel assay for Cdc14 specificity profiling. (a) Nomenclature and sequences of the 15 synthetic phosphopeptides used to assess conservation of Cdc14 specificity in plant pathogenic fungi. The peptides are grouped by colors representing alterations to different determinants of ScCdc14 specificity: black, phosphoamino acid identity; cyan, + 1 Pro; green, + 3 Lys/Arg; magenta, additional basic amino acids around + 3. The orange peptide assessed the negative impact of Pro at + 2. (b) Linear dynamic range of the LTQ Orbitrap Velos Pro mass spectrometer for measuring phosphopeptide desphorphorylation by Cdc14. Mean integrated LC–MS peak intensities for the 15 phosphopeptides in (a) were plotted as a function of the injected amount of each peptide. For all specificity constant measurements, 750 fmol each peptide were injected. (c) Example of LC–MS chromatograms extracted with Skyline over a reaction time course with the synthetic phosphopeptide pool from (a). Signals for the phosphorylated and dephosphorylated forms of the pS and + 3R peptides are shown at 3 different reaction times. Integrated peak areas are used to calculated k_cat/K_M as described in “Methods”. (d) Peak areas from data similar to (c) for the pS and + 3R peptides were used to calculate the fraction of substrate and product at different reaction times. Colors and lines match the peptide species from (c). Data were fit with a standard exponential reaction progress function, demonstrating that the MS assay accurately reflects expected enzyme reaction progress kinetics.

Figure 4

Substrate specificity is highly conserved in plant pathogenic fungal Cdc14 homologs. (a) The mean specificity constant, or k_cat/K_M, for the 8 indicated Cdc14 enzymes towards each of the 15 phosphopeptide substrates. The median value for each peptide is indicated with a short line. All data represent the average of 3 independent measurements. All k_cat/K_M values with errors are reported in Table 2. Due to the limited concentration of purified PsCdc14 we could not complete its analysis under identical conditions. However, a specificity profile for PsCdc14 at a single enzyme concentration is shown in Supplementary Figure S4 online. (b) The data from (a) are shown in line graph format to better illustrate the degree of overlap in specificity. (c) To validate the full phosphopeptide collection and highlight the range of Cdc14 activities, the median Cdc14 specificity constants from (a) (units M⁻¹ s⁻¹) were compared to related values using the broad specificity lambda protein phosphatase. Since enzyme concentration was not available for the commercial lambda phosphatase, true k_cat/K_M could not be calculated. Instead, lambda phosphatase values were normalized such that the maximal lambda and Cdc14 rates were equivalent.

Figure 5

Fungal Cdc14 homologs can be specifically inhibited by a substrate mimetic. (a) Structure of the synthetic peptide used for inhibition assays (sequence Glu-Val-pCF₂Ser-Pro-Thr-Lys-Arg-amide). (b) Dose response with the pCF₂Ser peptide from A and the indicated fungal Cdc14 enzymes using DiFMUP as substrate. Data represent the average of 5 or 6 independent trials. It was not practical to show error bars on the graph, however error values for replicate K_i measurements are provided in (c). Best fit lines were generated with a standard slope dose response function with plateaus set at 100% and 0% in Graphpad Prism. We did not have enough pCF₂Ser peptide to perform the full analysis on all 8 plant fungal pathogen homologs. (c) Each individual trial from the experiment in (b) was fit as described in b to generate IC₅₀, from which K_i was calculated (see “Methods”). Values represent the mean ± standard deviation. (d) Comparison of pCF₂Ser peptide inhibition of human tyrosine phosphatase PTP1B and dual specificity phosphatase VHR to ScCdc14 and human Cdc14A using DiFMUP at the measured K_M for each enzyme. Percent activity relative to a no inhibitor control was calculated and plotted. Data are means of 3 independent experiments and error bars are standard deviations. Numbers over the bars are p values from a t-test (unpaired, one-tail) comparing 0 and 200 µM inhibitor data.