Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2

Int J Biochem Cell Biol. 2018 Apr:97:52-61. doi: 10.1016/j.biocel.2018.02.003. Epub 2018 Feb 5.

Abstract

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.

Keywords: Abscisic acid (ABA); Binding affinity; Computational studies; Human lanthionine synthetase component C-like protein 2 (LANCL2); Site-directed mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Abscisic Acid / chemistry*
  • Abscisic Acid / metabolism
  • Amino Acid Substitution
  • Binding Sites
  • Humans
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphate-Binding Proteins
  • Recombinant Proteins
  • Surface Plasmon Resonance

Substances

  • LANCL2 protein, human
  • Membrane Proteins
  • Nuclear Proteins
  • Phosphate-Binding Proteins
  • Recombinant Proteins
  • Abscisic Acid