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

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. Iin this work, 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 $$α$$5 and $$α$$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 $$α$$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.

Authors:

Wang, Huanchen ^[1]; Gu, Chunfang ^[1]; Rolfes, Ronda J. ^[2]; Jessen, Henning J. ^[3]; Shears, Stephen B. ^[1]

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+ Show Author Affiliations

1. National Inst. of Health, Research Triangle Park, NC (United States). National Inst. of Environmental Health Sciences (NIEHS)
2. Georgetown Univ., Washington, DC (United States)
3. Albert Ludwigs Univ., Freiburg (Germany)

Publication Date:

Wed Mar 14 00:00:00 EDT 2018

Research Org.:

Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Org.:

National Institutes of Health (NIH), National Institutes of Environmental Health Sciences; Georgetown University Pilot Grant

OSTI Identifier:

1439638

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 293; Journal Issue: 18; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; inositol phosphate; crystal structure; enzyme mechanism; phosphatase; dual-specificity phosphoprotein phosphatase