Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Abstract

Inorganic polyphosphates (polyPs) are long polymers of orthophosphate units (Pi), linked by energy-rich phosphoanhydride bonds. Conserved histidine -helical (CHAD) domains of unknown biochemical function are often located at the C-terminus of polyP-metabolizing triphosphate tunnel metalloenzymes (TTMs), or can be found as stand-alone proteins in bacterial operons harboring polyP kinases or phosphatases. Here we report that bacterial, archaeal and eukaryotic CHAD domains are specific polyP binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central cavity surrounded by two conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano-to mid-micromolar range, but not DNA or other Pi-containing ligands. A 2.1 [A] CHAD - polyP complex structure reveals the phosphate polymer binding across a central pore and along the two basic patches. Mutational analysis of CHAD - polyP interface residues validates the complex structure and reveals that CHAD domains evolved to bind long-chain polyPs. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. In plants, CHAD domains bind polyP in vivo and localize to the nucleus and nucleolus, suggesting that plants harbor polyP stores in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.\n\nSignificanceA domain of unknown function termed CHAD, present in all kingdoms of life, is characterized as a specific inorganic polyphosphate binding domain. The small size of the domain and its high specificity for inorganic polyphosphates suggest that it could be used as a tool to locate inorganic polyphosphate stores in pro- and eukaryotic cells and tissues.