Multiple substrate recognition by yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase through phosphate clamping

Abstract

The yeast diadenosine and diphosphoinositol polyphosphate phosphohydrolase DDP1 is a Nudix enzyme with pyrophosphatase activity on diphosphoinositides, dinucleotides, and polyphosphates. These substrates bind to diverse protein targets and participate in signaling and metabolism, being essential for energy and phosphate homeostasis, ATPase pump regulation, or protein phosphorylation. An exhaustive structural study of DDP1 in complex with multiple ligands related to its three diverse substrate classes is reported. This allowed full characterization of the DDP1 active site depicting the molecular basis for endowing multisubstrate abilities to a Nudix enzyme, driven by phosphate anchoring following a defined path. This study, combined with multiple enzyme variants, reveals the different substrate binding modes, preferences, and selection. Our findings expand current knowledge on this important structural superfamily with implications extending beyond inositide research. This work represents a valuable tool for inhibitor/substrate design for ScDDP1 and orthologs as potential targets to address fungal infections and other health concerns.

Fig. 1. ScDDP1 structure.

(A) ScDDP1 cartoon representation with transparent surface (left) and topology diagram (right) showing α helices (brown), β strands (teal), and loops (cream). The specific insertion named as “nose” is marked as a dashed circle and darker surface. (B) Alignment between human and yeast orthologs of DDP1.Top row indicates the secondary structure of ScDDP1. The Nudix consensus and sequence in MutT1, a prototype of Ap₅A hydrolases, are shown at the bottom. Conserved residues and the nose are also highlighted. (C) Structural superposition of Nudix motifs in ScDDP1 and its orthologs HsDIPP1 and MsMutT1. (D) ScDDP1Δnose cartoon representation highlighting the remaining residues in the nose region as cyan sticks (left) and comparison between ScDDP1 and ScDDP1Δnose relative pyrophosphatase activity at 30 min. The data correspond to triplicates (right). WT, wild type.

Fig. 2. DDP1 inositide recognition.

(A) Chemical structures of InsPs and their analogues used in this work. (B) The five different binding modes (bms) detected for the inositides. InsP₆ is included in all bms as a reference. (C) Structure of ScDDP1:InsP₆ complex. Protein is shown in cartoons (Nudix region in brown), InsP₆ in sticks (C, O, and P atoms in teal, red, and orange, respectively), protein residues in lemon sticks (light orange from Nudix region) and hydrogen bonds in dashed lines. (D) Structure of ScDDP1:PCP-InsP₇ complex. Red and green spheres show water and Mg²⁺ ions, respectively. (E) Structure of ScDDP1:5-InsP₇ complex. (F) Structure of ScDDP1:PCP-InsP₈ complex. Bottom panels show the positions for phosphate recognition: six in InsP₆, PCP-InsP₇ and 5-InsP₇, whereas only five in PCP-InsP₈. Numbering of ScDDP1/HsDIPP1 residues is in black/green respectively. Isoform-specific residues are in squares and Mg2/Mg3 positions are inferred from HsDIPP1:5-InsP₇ complex. (G) Mg²⁺ interactions in ScDDP1:PCP-InsP₇ (left), HsDIPP1:5-InsP₇ (middle), and PCP:InsP₈ (right) complexes. (H) (Top) Pyrophosphatase activity (time = 30 min) of wt-ScDDP1, mutants, and wt-ScDDP1 in the presence of Fluor (pH 8) and wt-ScDDP1 (pH 4.5). Data correspond to triplicates. (Bottom) Structural superposition of wt-ScDDP1 (green) and ScDDP1-E80Q (orange) in the presence of 1-InsP₇. Mg3 is only present in wt-ScDDP1. (I) Structural alignment between ScDDP1 and HsDIPP isoforms. The structural secondary elements for ScDDP1 are specified.

Fig. 3. ScDDP1 binding of polyphosphates and dinucleotides.

(A) Structure of ScDDP1:Ap₅ complex. ScDDP1 is shown in yellow cartoons, its residues in teal sticks, the Ap₅ moiety in dark blue, and Mg²⁺ and water as green and red spheres, respectively; residue numbering in ScDDP1/HsDIPP1 in black/gray, respectively; the three specific residues of HsDIPP1 as white sticks; and hydrogen bonds as dashed lines. (B) Structure of ScDDP1:AMP-PNP complex. Two interacting molecules of AMP-PNP are shown as blue sticks. (C) Structure of ScDDP1:polyP15 complex. PolyP15 is shown as orange sticks. (D) Surface representation of ScDDP1 (orange) and HsDIPP1 (gray) showing different ligands: inositide (left, cyan), Ap₅ (middle, blue), and polyP15 (right, orange). The structures of HsDIPP1 with Ap₅ and polyP15 have been obtained by structural superposition with ScDDP1 complexes. For clarity, the nose is omitted from some panels. (E) Structural alignment between ScDDP1 and MsMutT1 enzymes highlighting residues involved in ligands binding. The elements on top represent ScDDP1 secondary structure. (F) Distinctive elements in nucleotide binding between ScDDP1 (left) and MsMutT1 (right). (G) Surface representation of ScDDP1 (orange) and MsMutT1 (green). The active site delineates a very different cavity to bind PCP-InsP₇, (cyan) Ap₅ (blue) or polyP15 (orange) in ScDDP1 and Ap₅ (blue) in MsMutT1. In MsMutT1, an InsP (cyan) has been placed to show the impossibility of its accommodation.

Fig. 4. wt-DDP1 and DDP1 mutants analysis.

Comparison of inflection temperatures (T_i) obtained by measuring the fluorescence 350/330-nm ratio over a range of temperatures of (A). Left: ScDDP1 at different pHs in free state and in HMP and InsP₆ bound forms. Right: ScDDP1 in the presence PCP-InsP₇ at different pHs in absence and presence of 5 mM MgCl₂. (B) Left: wt-DDP1 unbound and with the three types of ligands, represented by PCP-InsP₇ (inositides), Ap₅A and AMP-PNP (adenine-based), and HMP (polyPs) at pH 4.5 and pH 8.0. Right: Analysis with several concentrations of Ap₅A (100 μM to 1 mM) at pH 4.5. (C) DDP1 in complex with various inositide-based ligands at pH 4.5. (D) wt-DDP1 and mutants at pH 4.5 in the absence (top) and presence (bottom) of HMP, InsP₆, Ap₅A, and AMP-PNP. Difference inflection temperatures (ΔT_i) have been calculated between each mutant and wild type in absence of ligand as reference (top) or between each sample (either wt or mutant) in the presence of the ligand indicated and its free state (bottom). (E) wt-ScDDP1 and ScDDP1 mutants’ pyrophosphatase activity after 30 min. The T_i values and activity data correspond to triplicates. Error bars show the SDs (not visible when SD ≤ 0.1). When unspecified, ligand concentrations are 100 μM, except for Ap₅A and AMP-PNP that are 750 μM.