Format

Send to

Choose Destination
J Biol Chem. 2018 May 4;293(18):6905-6914. doi: 10.1074/jbc.RA117.001670. Epub 2018 Mar 14.

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

Author information

1
From the Inositol Signaling Group, Signal Transduction Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, huanchen.wang@nih.gov.
2
From the Inositol Signaling Group, Signal Transduction Laboratory, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709.
3
Department of Biology, Georgetown University, Washington, D. C. 20057, and.
4
Institute of Organic Chemistry, Albert Ludwigs University, Freiburg, 79104 Freiburg, Germany.

Abstract

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) 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 CX5R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the α5 and α6 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 α5 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.

KEYWORDS:

crystal structure; dual-specificity phosphoprotein phosphatase; enzyme mechanism; inositol phosphate; phosphatase

PMID:
29540476
PMCID:
PMC5936820
DOI:
10.1074/jbc.RA117.001670
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center