One Scaffold, Two Conformations: The Ring-Flip of the Messenger InsP8 Occurs under Cytosolic Conditions

Abstract

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a “flipped” conformation with five axial substituents. Using 13C-labeled InsPs/PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment. Remarkably, the most highly phosphorylated messenger 1,5(PP)2-InsP4 (also termed InsP8) readily adopts both conformations at physiological conditions. Environmental factors—such as pH, metal cation composition, and temperature—strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP8 from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg2+ decreased the binding constant Kd of InsP8 to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-responsive molecular switch.

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

“…Previous studies (Gaugler et al, 2022; Wang et al, 2022) and our biochemical analysis reveal AtPFA-DSPs as specific inositol 5-pyrophosphate phosphatases. As in the case of yeast Siw14 (Steidle et al, 2016), 5-InsP 7 is the preferred in vitro substrate for AtPFA-DSP1 in the presence and absence of Mg 2+ ions (Wang et al, 2022; Kurz et al, 2023) (Figure 1A, B and Supplementary Figure 2). The MpPFA-DSP1 ortholog shares the substrate specificity and overall activity with the Arabidopsis enzyme (Figure 3B and Supplementary Figure 6).…”

“…Both enzymes do not require a metal co-factor for catalysis (Lonetti et al, 2011; Steidle et al, 2016). However, the conformational equilibrium of PP-InsPs can be modulated by metal cations (Kurz et al, 2023) and hence we performed enzyme assays in the presence and absence of MgCl 2 (Figure 1A, B). Taken together, AtPFA-DSP1 and AtNUDT17 are bona fide inositol pyrophosphate phosphatases.…”

“…The fungal PFA-DSPs ScSiw14 and SpSiw14 are metal-independent cysteine-dependent phosphatases capable of hydrolyzing 1-InsP 7 , 5-InsP 7 and 1,5-InsP 8 with a preference for 5-InsP 7 (Sanchez et al, 2023; Steidle et al, 2016; Wang et al, 2018). The preferred substrate of the five PFA-DSPs in Arabidopsis is 5-InsP 7 in the presence of Mg 2+ ions (Kurz et al, 2023) in vitro (Gaugler et al, 2022; Wang et al, 2022).…”

The Role of Inositol Hexakisphosphate Kinase in the Central Nervous System

“…Some of these compounds even further showed varying degrees of selectivity within IP6K isoforms [ 32 , 35 , 138 ]. Tools that differentiate the roles of IP7 and IP8 are also needed to more fully determine the individual roles of these two molecules [ 139 ].…”

Assigning the Absolute Configuration of Inositol Poly- and Pyrophosphates by NMR Using a Single Chiral Solvating Agent

“…Signals of the pyrophosphates are visible in the range of −8 to −12 ppm. The shifts are strongly dependent on the pH value and the counterions [36]. Another novel compound that was synthesized in the context of this study is 5-PP-1/3-InsP1 (28).…”