Structural and biochemical characterization of Siw14: A protein-tyrosine phosphatase fold that metabolizes inositol pyrophosphates

Huanchen Wang, Chunfang Gu, Ronda J. Rolfes, Henning J. Jessen, Stephen B. Shears
2018 Journal of Biological Chemistry  
Edited by Joseph M. Jez Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals, plants, and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution, reaction mechanisms, and regulation. Here, we describe the 2.35-Å-resolution structure of the catalytic core of Siw14, a 5-PP-InsP phosphatase from Saccharomyces cerevisiae and a member of the protein tyrosine-phosphatase (PTP)
more » ... e (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses, thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs, but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. (a) The catalytic P-loop with the CX 5 R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the ␣5 and ␣6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the ␣5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. (c) The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis. . 2 The abbreviations used are: PP-InsP, inositol pyrophosphate; DIPP, diphosphoinositol polyphosphate phosphohydrolase; DUSP, dual-specific protein-tyrosine phosphatase; InsP 6 , inositol hexakisphosphate; 1-InsP 7 , 1-diphosphoinositol 2,3,4,5,6-pentakisphosphate; 5-InsP 7 , 5-diphospho-inositol 1,2,3,4,6-pentakisphosphate; 1,5-InsP 8 , 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate; 5-PP-InsP 4 , 5-diphosphoinositol 1,3,4,6-tetrakisphosphate; PTP, protein-tyrosine phosphatase.
doi:10.1074/jbc.ra117.001670 pmid:29540476 fatcat:7i73tmn4gncfrn5xxd4fmnizya