Nup159 Weakens Gle1 Binding to Dbp5 But Does Not Accelerate ADP Release

J Mol Biol. 2018 Jul 6;430(14):2080-2095. doi: 10.1016/j.jmb.2018.05.025. Epub 2018 May 19.

Abstract

Dbp5, DDX19 in humans, is an essential DEAD-box protein involved in mRNA export, which has also been linked to other cellular processes, including rRNA export and translation. Dbp5 ATPase activity is regulated by several factors, including RNA, the nucleoporin proteins Nup159 and Gle1, and the endogenous small-molecule inositol hexakisphosphate (InsP6). To better understand how these factors modulate Dbp5 activity and how this modulation relates to in vivo RNA metabolism, a detailed characterization of the Dbp5 mechanochemical cycle in the presence of those regulators individually or together is necessary. In this study, we test the hypothesis that Nup159 controls the ADP-bound state of Dbp5. In addition, the contributions of Mg2+ to the kinetics and thermodynamics of ADP binding to Dbp5 were assessed. Using a solution based in vitro approach, Mg2+ was found to slow ADP and ATP release from Dbp5 and increased the overall ADP and ATP affinities, as observed with other NTPases. Furthermore, Nup159 did not accelerate ADP release, while Gle1 actually slowed ADP release independent of Mg2+. These findings are not consistent with Nup159 acting as a nucleotide exchange factor to promote ADP release and Dbp5 ATPase cycling. Instead, in the presence of Nup159, the interaction between Gle1 and ADP-bound Dbp5 was found to be reduced by ~18-fold, suggesting that Nup159 alters the Dbp5-Gle1 interaction to aid Gle1 release from Dbp5.

Keywords: DEAD-box protein; Dbp5; Gle1; Nup159; kinetics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Diphosphate / analogs & derivatives
  • Adenosine Diphosphate / metabolism*
  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • DEAD-box RNA Helicases / metabolism*
  • Humans
  • Magnesium / metabolism
  • Nuclear Pore Complex Proteins / metabolism*
  • Nucleocytoplasmic Transport Proteins / metabolism*
  • Protein Binding
  • RNA / metabolism
  • ortho-Aminobenzoates / metabolism

Substances

  • Gle1 protein, human
  • Nuclear Pore Complex Proteins
  • Nucleocytoplasmic Transport Proteins
  • ortho-Aminobenzoates
  • Adenosine Diphosphate
  • RNA
  • 3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate
  • 3'-O-(N-methylanthraniloyl) ATP
  • Adenosine Triphosphate
  • DDX19B protein, human
  • DEAD-box RNA Helicases
  • Magnesium