Phosphate transfer from inositol pyrophosphates InsP5PP and InsP4(PP)2: A semi-empirical investigation

doi:10.1016/j.bmcl.2006.09.066

Bioorganic & Medicinal Chemistry Letters

Volume 17, Issue 1, 1 January 2007, Pages 183-188

https://doi.org/10.1016/j.bmcl.2006.09.066 Get rights and content

Abstract

A novel phosphate transfer process involving the non-enzymatic transfer of a phosphate group from inositol pyrophosphates to serine residues in proteins has been recently reported. Semi-empirical calculations at the PM3/SM5.2 level were undertaken to explore the effect of inositol pyrophosphate structure and overall charge on the thermodynamics of this phosphate transfer.

Graphical abstract

PM3/SM5.2 semi-empirical calculations were used to calculate the free energy of phosphate transfer from InsP₅PP and InsP₄(PP)₂ to methanol as a model system for non-enzymatic phosphorylation of protein residues by these intracellular pyrophosphates.

Section snippets

Acknowledgments

The authors thank NSERC Canada and the University of Waterloo for financial support.

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Inositol pyrophosphates (PP-InsPs) comprise an evolutionarily ancient family that exhibit many regulatory roles throughout the eukaryotic kingdoms. These molecules have the extraordinary feature of possessing the most concentrated three-dimensional array of phosphate groups in Nature. This characteristic endows the PP-InsPs with a palette of physicochemical properties, each of which can be separately called upon to serve a wide range of signaling situations. In this review we will illustrate how this unusual, mechanistic flexibility has a consistent functional theme that PP-InsPs regulate multiple aspects of bioenergetic and metabolic homeostasis, in animals, plants and fungi. We will also describe the small-molecule kinases and phosphatases that are responsible for switching on and off PP-InsP signals; these enzymes have a number of rare properties that can finely tune PP-InsP turnover in response to changing environmental conditions. Finally, we will draw attention to important uncertainties in this wide-ranging field of PP-InsP research that remain to be resolved.
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However, electrostatic and steric repulsion from adjacent monophosphates is likely to increase the thermodynamic favourability of β-phosphate release (Hand and Honek, 2007). Indeed, this phosphate release is expected to be strongly exothermic, with semi-empirical calculations revealing that the free enthalpy (ΔH) for transfer of the β-phosphate from 5-IP7 to methanol can reach −38.3 kcal/mol at pH 6.8 (Hand and Honek, 2007). As discussed above, during the pyrophosphorylation reaction, the Asp/Glu-rich disordered substrate peptide must associate with the Mg2+-IP7 complex via coordinate bond formation.
Inositol pyrophosphates (PP-IPs) are a class of energy rich metabolites present in all eukaryotic cells. The hydroxyl groups on these water soluble derivatives of inositol are substituted with diphosphate and monophosphate moieties. Since the discovery of PP-IPs in the early 1990s, enormous progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways – allosteric regulation by direct binding, or post-translational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg²⁺-dependent, but enzyme independent transfer of a β-phosphate from a PP-IP to a pre-phosphorylated serine residue located in an acidic motif, within an intrinsically disordered protein sequence. This distinctive post-translational modification has been shown to regulate diverse cellular processes, including rRNA synthesis, glycolysis, and vesicle transport. However, our understanding of the molecular details of this phosphotransfer from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. This review discusses our current knowledge of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet overlooked post-translational modification.
Dynamics of Substrate Processing by PPIP5K2, a Versatile Catalytic Machine
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Citation Excerpt :
The X-ray structure of PPIP5K2 in complex with ADP and 1,5-IP8 (PDB: 4Q4C) was used to build the starting configuration for eight independent MD runs (1 μs each). The activation barrier for hydrolysis of the 1-β-phosphate of IP8 is not known, although it is generally considered to be less than that for ATP (Hand and Honek, 2007). Indeed, the ability of PP-IPs to pyrophosphorylate proteins by a mechanism that is entirely chemical in nature is consistent with a low activation barrier for their phosphoanhydride bond cleavage (Saiardi et al., 2004).
Processing of substrates by enzymes can only be fully understood through their conformational dynamics; this is particularly true for the diphosphoinositol pentakisphosphate kinase PPIP5K2, an enzyme with critical roles in cell signaling and bioenergetic homeostasis. PPIP5K2 is remarkable for the reversible nature of its kinase activity, its unique ligand-stimulated ATPase activity, and the substrate traveling between two ligand-binding sites. Here we use molecular dynamics and data analysis techniques to rationalize these PPIP5K2 activities, thereby increasing our understanding of complex enzymatic mechanisms. In particular, we demonstrate how the enzyme's distinctive, ratchet-like mechanism harnesses the energy of random fluctuations to significantly reduce the entropy toll for intramolecular substrate transfer. We show that pre-reaction pulling forces along the reaction coordinate are predictive of the various PPIP5K2 catalytic activities. An unexpected possibility, raised by these computational studies, that 3,5-IP8 might be a substrate for dephosphorylation was experimentally interrogated and confirmed in a luciferase assay.
Inositol phosphate kinases: Expanding the biological significance of the universal core of the protein kinase fold
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Citation Excerpt :
As for IP6Ks, these are of particular interest because they add a 5-β-phosphate to InsP6, yielding 5-InsP7, an ‘inositol pyrophosphate’ (Saiardi et al., 1999; Shears, 2017). The latter is an ‘energetic’ (Hand and Honek, 2007), multifunctional molecule that receives particular attention for its key roles in bioenergetic homeostasis, cancer, and aging (Chakraborty, 2017; Shears, 2017). Bearing in mind the enormous significance of the PDKG-InsPK family, it is unsurprising that several groups have taken a structural approach to characterizing these enzymes (Bennett et al., 2006; Chamberlain et al., 2005; Endo-Streeter et al., 2012; Gonzalez et al., 2004; Holmes and Jogl, 2006; Miller and Hurley, 2004; Wang et al., 2014; Wang and Shears, 2017).
The protein kinase family is characterized by substantial conservation of architectural elements that are required for both ATP binding and phosphotransferase activity. Many of these structural features have also been identified in homologous enzymes that phosphorylate a variety of alternative, non-protein substrates. A comparative structural analysis of these different kinase sub-classes is a portal to a greater understanding of reaction mechanisms, enzyme regulation, inhibitor-development strategies, and superfamily-level evolutionary relationships. To serve such advances, we review structural elements of the protein kinase fold that are conserved in the subfamily of inositol phosphate kinases (InsPKs) that share a PxxxDxKxG catalytic signature: inositol 1,4,5-trisphosphate kinase (IP3K), inositol hexakisphosphate kinase (IP6K), and inositol polyphosphate multikinase (IPMK). We describe conservation of the fundamental two-lobe kinase architecture: an N-lobe constructed upon an anti-parallel β-strand scaffold, which is coupled to a largely helical C-lobe by a single, adenine-binding hinge. This equivalency also includes a G-loop that embraces the β/γ-phosphates of ATP, a transition-state stabilizing residue (Lys/His), and a Mg-positioning aspartate residue within a catalytic triad. Furthermore, we expand this list of conserved structural features to include some not previously identified in InsPKs: a ‘gatekeeper’ residue in the N-lobe, and an ‘αF’-like helix in the C-lobe that anchors two structurally-stabilizing, hydrophobic spines, formed from non-consecutive residues that span the two lobes. We describe how this wide-ranging structural homology can be exploited to develop lead inhibitors of IP6K and IPMK, by using strategies similar to those that have generated ATP-competing inhibitors of protein-kinases. We provide several examples to illustrate how such an approach could benefit human health.
The significance of the 1-kinase/1-phosphatase activities of the PPIP5K family
2017, Advances in Biological Regulation
Citation Excerpt :
Inositol pyrophosphates (PP-InsPs; Fig. 1) are a specialized class of cell signaling molecules that boast a unique, functionally significant feature: the most crowded three-dimensional arrangement of phosphate groups that has been observed throughout Nature. Crammed around a six-carbon inositol scaffold are as many as seven or eight phosphates (for “InsP7” and “InsP8” respectively, see Fig. 1), including diphosphate groups that are highly ‘energetic’ (Hand and Honek, 2007; Shears, 2015). Yeasts and metazoan cells can phosphorylate InsP6 to InsP8 through two parallel pathways (Fig. 1), which utilize two separate classes of enzymes to form diphosphate groups: the 5-kinases (the IP6Ks (E.C.2.7.4.21); Draskovic et al., 2008; Saiardi et al., 1999) and the 1-kinases (the PPIP5Ks (E.C. 2.7.4.24); Lin et al., 2009; Wang et al., 2012).
The inositol pyrophosphates (diphosphoinositol polyphosphates), which include 1-InsP₇, 5-InsP₇, and InsP₈, are highly ‘energetic’ signaling molecules that play important roles in many cellular processes, particularly with regards to phosphate and bioenergetic homeostasis. Two classes of kinases synthesize the PP-InsPs: IP6Ks and PPIP5Ks. The significance of the IP6Ks - and their 5-InsP₇ product - has been widely reported. However, relatively little is known about the biological significance of the PPIP5Ks. The purpose of this review is to provide an update on developments in our understanding of key features of the PPIP5Ks, which we believe strengthens the hypothesis that their catalytic activities serve important cellular functions. Central to this discussion is the recent discovery that the PPIP5K is a rare example of a single protein that catalyzes a kinase/phosphatase futile cycle.

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Phosphate transfer from inositol pyrophosphates InsP5PP and InsP4(PP)2: A semi-empirical investigation

Abstract

Graphical abstract

Section snippets

Acknowledgments

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Curr. Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Mol. Catal. A: Chem.

Tetrahedron Lett.

Chem. Phys. Lett.

J. Inorg. Biochem.

J. Biol. Chem.

Carbohydr. Res.

Carbohydr. Res.

Biochemistry

Biochem. J.

J. Mol. Evol.

Annu. Rev. Biochem.

Biochem. J.

Biochem. J.

Proc. Natl. Acad. Sci. U.S.A.

Biochem. J.

Proc. Natl. Acad. Sci. U.S.A.

Proc. Natl. Acad. Sci. U.S.A.

Science

J. Am. Chem. Soc.

J. Phys. Chem. B

Phosphate transfer from inositol pyrophosphates InsP₅PP and InsP₄(PP)₂: A semi-empirical investigation