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