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.

Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms

“…2B). Because the InsPs have a high propensity for coordinating metal cations, and in particular form strong complexes with magnesium ions (42)(43)(44), magnesium concentrations were kept constant at 1 mM (corresponding to the approximate concentration of freely available cellular Mg 2+ ) during affinity enrichment, wash steps, and elution. The eluted fractions were resolved by SDS/PAGE, and the gel lanes were excised.…”

“…Consequently, we repeated the affinity enrichments using "metal-free" conditions, where lysis, washing, and elution buffers contained 1 mM EDTA. The metal chelator will remove divalent and trivalent cations, which interact tightly with the highly phosphorylated inositols (44,45). Addition of EDTA significantly altered the protein interactions of reagents 1 and 2, and the majority of the 82 proteins that were isolated with EDTA present had not been enriched in the presence of magnesium ions (Fig.…”

“…Identification of several known InsP-and PP-InsP-interacting partners confirmed the efficiency of the affinity reagents under conditions controlling metal cation availability. However, it should be noted that some of these InsP-protein interactions may be more transient in a cellular setting, where InsPs are likely to form strong complexes with magnesium (43,44).…”

Metabolism and Functions of Inositol Pyrophosphates: Insights Gained from the Application of Synthetic Analogues

“…Potter’s group replaced the bisphosphate of 5-InsP 7 with phosphonoacetate (PA) [ 23 ]. Subsequently, the PA- analog of 1,5-InsP 8 and PCP-analogues of 1-InsP 7 and 1,5-InsP 8 were also synthesized [ 25 , 26 , 27 ].…”

“…This observation raises the possibility that the product escapes from the catalytic pocket via the same capture site. In contrast, in corresponding crystal complexes that contain either 1,5-PCP-InsP 8 or 5-PCP-InsP 7 , these ligands were only observed in the catalytic pocket [ 25 , 27 ], just as is the case with the natural substrates [ 30 , 34 ]. Nevertheless, the PA-version was more informative because it identified the capture site.…”