New Structural Insights Reveal an Expanded Reaction Cycle for Inositol Pyrophosphate Hydrolysis by Human DIPP1

Abstract

Nudix hydrolases, an enzyme superfamily with representatives in all classes of organisms, possess a conserved catalytic sequence (Nudix motif), GX5EX7REUXEEXGU, where ‘U’ designates a hydrophobic residue. The Diphosphoinositol Polyphosphate Phosphohydrolases (DIPPs) represent one subgroup of the Nudix family. By metabolizing diphosphoinositol polyphosphates (PP-InsPs), DIPPs switch-off their signaling activities that regulate metabolic homeostasis. However, there has not been a molecular rationalization of DIPPs catalytic mechanism, substrate recognition and reaction specificity. We now provide X-ray analysis of the catalytic core of DIPP1 in crystals complexed with either natural PP-InsPs, alternative PP-InsP stereoisomers, or non-hydrolysable methylene bisphosphonate analogs (“PCP-InsPs”). The conclusions that we draw from these data are interrogated by studying the impact upon catalytic activity upon mutagenesis of certain key residues. We present a picture of a V-shaped catalytic furrow with overhanging ridges constructed from flexible positively charged side chains; within this cavity, the labile phosphoanhydride bond is appropriately positioned at the catalytic site by an extensive series of interlocking polar contacts which we analogize as “suspension cables.” We demonstrate functionality for a triglycine peptide within a β-strand which represents a non-canonical addition to the standard Nudix catalytic core structure. We describe pre-reaction enzyme/substrate states which we posit to reflect a role for electrostatic steering in substrate capture. Finally, through time-resolved analysis, we uncover a chronological sequence of DIPP1/product post-reaction states, one of which may rationalize a role for InsP6 as an inhibitor of catalytic activity. We have gained considerable structure-based insight into the reaction cycle for DIPP1-catalyzed PP-InsP hydrolysis and can use this information to design inhibitors of an enzyme with proposed roles in obesity, insulin signaling and sarcopenia.