Biochemical and structural characterization of an inositol pyrophosphate kinase from a giant virus

Abstract

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 & Aring;. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the gamma-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches. Inositol phosphate kinases regulate many biological processes in eukaryotes, but recent discoveries of archaeal homologs suggest an ancient evolutionary origin. Here, characterization of a primordial ATP-dependent inositol pyrophosphate kinase encoded by the giant virus genome of Terrestrivirus provides insights into the evolution of inositol phosphate kinases.Terrestrivirus TvIPK specifically pyrophosphorylates a range of myo- and scyllo-based inositol polyphosphates both in vitro and when ectopically expressed in yeast. X-ray crystallographic analysis of TvIPK in complex with its substrates or products reveals conserved catalytic and architectural features. Positively charged and flexible side chains allow recognition of the diverse range of substrates. Pyrophosphorylation depends on ATP gamma-phosphate orientation via an Arg residues in a conserved "G-loop." TvIPK, an ATP-dependent inositol pyrophosphate kinase from Terrestrivirus, a putative member of the Mimiviridae family of giant viruses, offers insights into the evolution of inositol phosphate signaling.