Substrate recognition and mechanism revealed by ligand-bound polyphosphate kinase 2 structures

Abstract

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg²⁺ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.

Keywords: enzyme structure; kinase; kinetics; polyphosphate.

Fig. 1.

PPK2 catalysis and substrate binding. (A) Phosphotransfer reactions catalyzed by PPK2 classes. R, nucleobase. (B) PolyP binding to FtPPK2. The lid loop is shown in yellow and the Walker A motif (P-loop) is shown in blue. P9, nonaphosphate. (C) Active-site region of the FtPPK2:AMPPCPPP:polyP complex. The lid loop is shown in yellow. Ad, adenine moiety; P6, hexaphosphate; W, water.

Fig. 2.

PPK2 nucleotide-binding. (A) Overlay of MrPPK2:ADP:PP_i complex (blue) with the FtPPK2:AMPPCPPP:polyP complex (green). (B) Detail of nucleotide interactions for MrPPK2. (C) Detail of nucleotide interactions for FtPPK2. Magnesium ions shown as larger spheres, water molecules as smaller red spheres. Nuc, AMPPCPPP nucleotide. (D) Overlay of bound nucleotide conformations derived from the MrPPK2:ADP:PP_i complex (blue) and the FtPPK2:AMPPCPPP:polyP complex (green).

Fig. 3.

PPK2 mechanism and interaction with polyP. (A) Proposed mechanism of FtPPK2 and the modified nucleotide-binding mode for MrPPK2 that permits phosphorylation of the α-phosphate (highlighted in red). Blue curly arrows indicate the forward reaction (ATP formation), red curly arrows the reverse reaction, and black curly arrows are common to both. (B) Structure of FtPPK2 Asp117Asn variant cocrystallized with polyP. Labels indicate the polyP lengths: P6, P11, and P24.