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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.

SUBMITTER: Parnell AE

PROVIDER: S-EPMC5879653 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Parnell Alice E AE Mordhorst Silja S Kemper Florian F Giurrandino Mariacarmela M Prince Josh P JP Schwarzer Nikola J NJ Hofer Alexandre A Wohlwend Daniel D Jessen Henning J HJ Gerhardt Stefan S Einsle Oliver O Oyston Petra C F PCF Andexer Jennifer N JN Roach Peter L PL

Proceedings of the National Academy of Sciences of the United States of America 20180312 13

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 bot ...[more]

PMID: 29531036

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Project description:Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg2+ or Mn2+; IP6, 5-IP7, or 1,5-IP8) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP6 or 5-IP7 engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N6. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N6-benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP6, 5-IP7, and 1,5-IP8. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.

Dataset Information

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

Publications

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

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