Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues

Abstract

The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.

Keywords: biological activity; conformation analysis; phosphorylation; protonation; signal transduction.

Figure 1

Inositol pyrophosphates and their corresponding bisphosphonate analogues. a) Abbreviated biosynthetic pathway to generate the messengers 5-diphosphoinositol pentakisphosphate (5PP-InsP₅), and 1,5-bis(diphosphoinositol) tetrakisphosphate (1,5(PP)₂-InsP₄). b) Bisphosphonate analogues reported here, and their applications.

Figure 2

Structural analysis of bisphosphonate analogues in complex with PPIP5K2. a) Electrostatic surface presentation of the active site of PPIP5K2 with 5PCP-InsP₅ bound. b) Superimposition of the catalytic domains of PPIP5K2 bound to 5PCP-InsP₅ (cyan C–C bonds) or 5PP-InsP₅ (grey C–C bonds and white spheres for all atoms). c) Superimposition of the carbon atoms of the inositol ring in 5PCP-InsP₅ (cyan C–C bonds) or 5PP-InsP₅ (grey C–C bonds and white spheres for all atoms). d) Electrostatic surface presentation of the active site of PPIP5K2 with 3,5(PCP)₂-InsP₄ bound. e) Superimposition of the catalytic domains of PPIP5K2 bound to 3,5(PCP)₂-InsP₄ (yellow C–C bonds) or 3,5(PP)₂-InsP₄ (white C–C bonds and white spheres for all atoms). f) Superimposition of the carbon atoms of the inositol ring in 3,5(PCP)₂-InsP₄ (yellow C–C bonds) or 3,5(PP)₂-InsP₄ (white C–C bonds and white spheres for all atoms).

Figure 3

NMR titration of 5PCP-InsP₅ (1) without coordinating counter ions. a) and b) ³¹P NMR chemical shifts of 1 as a function of pH, measured in 0.15M NMe₄Cl at 22.0 °C. Solid lines indicate the chemical shifts predicted by HypNMR, according to the protonation constants given in Table S1. c) Corresponding species distribution diagram of 1. Predictions are for [L]=1.0 mM. The pH range of the ligand conformational change is highlighted in grey.

Figure 4

pH-Dependent conformational change of 5PCP-InsP₅ (1). a) Schematic illustrating the transition from the 1a5e (1 axial, 5 equatorial) to 5a1e (5 axial, 1 equatorial) conformation at elevated pH. b)–d) DFT optimised geometries for the most stable conformers of L¹³⁻ (b), HL¹²⁻ (c), and H₂L¹¹⁻ (d). The intramolecular hydrogen bonds are shown as black dotted lines. Phosphoryl groups forming intramolecular hydrogen bonds are in bold. Colour code: C (grey), H (white), O (red), P (orange).

Figure 5

Species distribution diagrams of 5PCP-InsP₅ (1) in the presence of potassium and magnesium ions. a) [1] =1.0 mm, [K⁺]=150 mM. b) [1] =1.0 mM, [K⁺]=150 mm, [Mg²⁺]=1.0 mm. T =22.08C. The pH range of the ligand conformational change is highlighted in grey.

Figure 6

DFT optimised geometries for the detected 5PCP-InsP₅ species in complex with potassium and magnesium ions. Intramolecular hydrogen bonds are shown as black dotted lines. Two unique hydrogen bonds from P5β to a water molecule in the first coordination sphere of magnesium are highlighted as green dotted lines. Phosphoryl groups forming intramolecular hydrogen bonds are in bold. Colour code: C (grey), H (white), O (red), P (orange), K (violet), Mg (lime green).

Figure 7

Effect of magnesium ions on the conformational equilibrium of InsP₆, 5PCP-InsP₅ (1) and rac-(PCP)₂-InsP₄ (rac-2). Schematic illustrating a shift of the conformational change of InsP₆, 1 and rac-2, towards physiological pD upon addition of magnesium.

Scheme 1

Synthesis of 5PCP-InsP₅ (1).

Scheme 2

Synthesis of 1,5-(PCP)₂-InsP₄ and 3,5-(PCP)₂-InsP₄.