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. 2011 Apr 14;472(7342):238-42.
doi: 10.1038/nature09862. Epub 2011 Mar 27.

A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export

Ben Montpetit  1 , Nathan D Thomsen, Kara J Helmke, Markus A Seeliger, James M Berger, Karsten Weis
Affiliations

A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export

Ben Montpetit et al. Nature. .

Abstract

Superfamily 1 and superfamily 2 RNA helicases are ubiquitous messenger-RNA-protein complex (mRNP) remodelling enzymes that have critical roles in all aspects of RNA metabolism. The superfamily 2 DEAD-box ATPase Dbp5 (human DDX19) functions in mRNA export and is thought to remodel mRNPs at the nuclear pore complex (NPC). Dbp5 is localized to the NPC via an interaction with Nup159 (NUP214 in vertebrates) and is locally activated there by Gle1 together with the small-molecule inositol hexakisphosphate (InsP(6)). Local activation of Dbp5 at the NPC by Gle1 is essential for mRNA export in vivo; however, the mechanistic role of Dbp5 in mRNP export is poorly understood and it is not known how Gle1(InsP6) and Nup159 regulate the activity of Dbp5. Here we report, from yeast, structures of Dbp5 in complex with Gle1(InsP6), Nup159/Gle1(InsP6) and RNA. These structures reveal that InsP(6) functions as a small-molecule tether for the Gle1-Dbp5 interaction. Surprisingly, the Gle1(InsP6)-Dbp5 complex is structurally similar to another DEAD-box ATPase complex essential for translation initiation, eIF4G-eIF4A, and we demonstrate that Gle1(InsP6) and eIF4G both activate their DEAD-box partner by stimulating RNA release. Furthermore, Gle1(InsP6) relieves Dbp5 autoregulation and cooperates with Nup159 in stabilizing an open Dbp5 intermediate that precludes RNA binding. These findings explain how Gle1(InsP6), Nup159 and Dbp5 collaborate in mRNA export and provide a general mechanism for DEAD-box ATPase regulation by Gle1/eIF4G-like activators.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. The Gle1IP6-Δ90Dbp5-ADP complex
a, Structure of Dbp5 bound to ADP (magenta spheres) and Gle1 (see colour key). b, Side view showing IP6 (coloured spheres) bound at the interface between Gle1 and Dbp5. c, Detailed view of the IP6 binding interface (see colour key). IP6 is shown as grey sticks with orange phosphate and red oxygen atoms. Nitrogen atoms are in dark blue. d, IP6 binding is required for maximal Dbp5 ATPase stimulation with RNA. Error bars represent s.d. (n=3). e, Structural superposition of the RNA-Δ90Dbp5 complex with Gle1IP6-Δ90Dbp5. Arrows depict large rigid body movement in Dbp5. f, Van der Waals surface view of the RNA-Δ90Dbp5 complex and the Gle1IP6-Δ90Dbp5 complex coloured by solvent accessible electrostatics. Abbreviations: kb – Boltzmann’s constant (Joules/Kelvin), T – temperature (310 Kelvin), ec – charge of an electron (1.602 x 10−19 Coulombs).
Figure 2
Figure 2. Comparison of Gle1IP6-Δ90Dbp5 and eIF4A-eIF4G
a, Structural superposition of Gle1IP6-Δ90Dbp5 with eIF4G-eIF4A and AMP (PDB ID: 2VSO) (see colour key). b, View of the C-terminal RecA-like domain binding interface. Unique α-helices present in both Dbp5 and Gle1 form the IP6 biding pocket (boxed in figure). c, d, Residues involved in the formation of the N-terminal RecA-like domain binding interface in (c) Gle1IP6-Δ90Dbp5 and (d) eIF4A-eIF4G. e, Measured ATPase activity using wild type or mutant Gle1. Error bars represent s.d. (n=3). f, g, RNA release from Dbp5E240Q or eIF4AE172Q monitored by fluorescence polarization. Representative curves are shown.
Figure 3
Figure 3. The Gle1IP6-Δ90Dbp5-Nup159 complex
a, Two views of the Gle1IP6-Dbp5-Nup159 complex (see colour key). b, The N-terminal RecA-like domain binding interface between Dbp5- Gle1IP6 is altered in the presence of Nup159 (compare to Fig. 2c). Residues involved in the binding interface (sticks) are labelled. c, Superposition of the N-terminal RecA-domain among the three structural states of Dbp5. Arrow highlights the movement of the CTD and catalytic arginine finger residues (sticks) among the three states (see colour key). For clarity, the Gle1-ADP intermediate is shown slightly transparent, and both the arginine finger side chains and ADP have been removed. d, Inhibition of the RNA stimulated ATPase activity of Dbp5 by Nup159 is overcome in the presence of Gle1. Error bars represent s.d. (n=3).
Figure 4
Figure 4. Model of the Dbp5 mechanochemical cycle
In the presence ATP, Dbp5 binds RNA causing local destabilization and remodelling of duplexed RNA or RNA-protein complexes (state I). ATP hydrolysis then allows the activator (Gle1) to bind both the C-terminal and N-terminal RecA-like domains, separating the two RecA-like domains and promoting RNA release (state II). For Dbp5 it is currently not known when Pi is released following hydrolysis, but this will likely occur prior to the formation of state II. Subsequent release of the bound RNA allows Nup159 to bind Dbp5 causing the two RecA domains to further separate (state III). The formation of this state could then facilitate ADP release, prevent rebinding of the RNA, and aid in enzyme recycling (state IV). Crystal structure model in state IV is PDB ID:3FHO. For colour coding details see key.
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