Positively-charged semi-tunnel is a structural and surface characteristic of polyphosphate-binding proteins: an in-silico study

Abstract

Phosphate is essential for all major life processes, especially energy metabolism and signal transduction. A linear phosphate polymer, polyphosphate (polyP), linked by high-energy phosphoanhydride bonds, can interact with various proteins, playing important roles as an energy source and regulatory factor. However, polyP-binding structures are largely unknown. Here we proposed a putative polyP binding site, a positively-charged semi-tunnel (PCST), identified by surface electrostatics analyses in polyP kinases (PPKs) and many other polyP-related proteins. We found that the PCSTs in varied proteins were folded in different secondary structure compositions. Molecular docking calculations revealed a significant value for binding affinity to polyP in PCST-containing proteins. Utilizing the PCST identified in the β subunit of PPK3, we predicted the potential polyP-binding domain of PPK3. The discovery of this feature facilitates future searches for polyP-binding proteins and discovery of the mechanisms for polyP-binding activities. This should greatly enhance the understanding of the many physiological functions of protein-bound polyP and the involvement of polyP and polyP-binding proteins in various human diseases.

Fig 1. Model structures of Positively-Charged Semi-Tunnel (PCST).

(A) PPK4 (PDB ID: 3G3Q); (B) PPK1 (PDB ID: 1XDP); (C) PPX/GPPA (PDB ID: 1T6C); (D) HsPLAP (PDB ID: 1EW2). Protein structure (left) and protein surface graphic (right). Colors on graphics represent surface charges of the 3D structure of the protein. Blue = positive; Red = negative. Yellow double end arrows indicate observed strip of PCST.

Fig 2. Binding affinities of polyP to polyP positive control proteins.

PPK4 (PDB ID: 3G3Q); PPK1 (PDB ID:1XDO);PPX/GPPA (PDB ID: 1T6C); HsPLAP (PDB ID: 1EW2). PolyP ligands included Ap₅ (A) and Ap₆A (B). Each circle, square, or triangle represented an estimated free energy of binding from the molecular docking calculations using Ap₅ or Ap₆A ligand and different polyP positive control proteins.

Fig 3. Predicted binding energies of polyP to PPK partial structural analogues with or without a Positively-Charged Semi-Tunnel.

Each circle or square represented an estimated free energy of binding from the molecular docking calculations using Ap₅ ligand and different PPK partial structural analogues.

Fig 4. PPK3 subunits and sequences.

(A) Multiple alignment of PPK3 subunit and actin amino acid sequences. Black arrows and blue highlights indicate strands; boxes and purple highlights indicate helices; yellow highlights indicate turns; inverted triangles and green highlights indicate nucleotide-binding regions. (B) The overlap of the backbone structures of the PPK3 subunits. α(blue); β(purple); ξ(green).

Fig 5. Binding affinities of polyP to subunits of PPK3.

Each circle, square, or triangle represented an estimated free energy of binding from the molecular docking calculations using Ap5 ligand and different PPK3 subunit.

Fig 6. PPK3 3D structure of subunits.

(A) β subunit; (B) ξ subunit; (C) α subunit. Protein structure (left) and protein surface graphic (right). Colors on graphics represent surface charges of the 3D structure of the protein. Blue = positive; Red = negative. Yellow double end arrows indicate observed strip of PCST.