Structural basis for the function of DEAH helicases

Abstract

DEAH helicases participate in pre-messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p-ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide-binding motif. A beta-hairpin from the second RecA domain is wedged between two carboxy-terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C-terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.

Figure 1

The structure of Prp43p. (A) Representation of Prp43p bound to ADP in two orientations differing by a 180° rotation. The extended NTD region is coloured blue, RecA-1 green, RecA-2 magenta, the 5′HP yellow, the WHD orange, the ratchet domain cyan and the CTD red. A long helix in the ratchet domain, containing three conserved arginines potentially involved in RNA interaction, is labelled ‘3R'. (B) Prp43p sequence conservation, based on supplementary Fig S2 online, mapped on the molecular surface in the same orientations as in (A). The 5′HP surface is outlined in yellow in the right panel. (C) Close-up of the interaction between the 5′HP and the WHD. Hydrogen bonds and salt bridges are shown as dashed lines. The orientation is the same as in (A), but the focus is behind the ratchet domain. (D) Predicted movement of the 5′HP between the ADP state (yellow) and a modelled ATP state (grey). The orientation is rotated −120° about the vertical axis with respect to the right panel of (A). C, conserved; CTD, carboxy-terminal domain; NC, non-conserved; NSD, not sufficient data; NTD, amino-terminal domain; SC, strictly conserved; WHD, winged-helix domain.

Figure 2

The ADP binding site. (A) The DEAH-specific binding site for the adenine base between Arg 159 in RecA-1 and Phe357 in RecA-2. Water molecules are shown as red spheres. (B) Comparison of the nucleotide binding sites of a DEAH-box and DEAD-box (grey) helicase (Andersen et al, 2006). (C) Schematic representation of direct interactions between the ADP molecule and Prp43p. Dashed lines represent backbone interactions (eletrostatic or hydrogen bonds) and full lines represent side-chain interactions. The different motifs are shown in bold. (D) The interactions between ADPNP and eIF4AIII (DEAD-box helicase) are shown as in (C). The DEAD-box specific Q motif is responsible for direct recognition of adenine in this class of helicases.

Figure 3

RNA binding to Prp43p. (A) Surface representation of Prp43p with the 5′HP present (left) and removed (right) to show its occlusion of a pocket between RecA-1, RecA-2 and the ratchet domains expected to bind ssRNA in the ATP state. An RNA placed by comparison with the exon junction complex (Research Collaboratory for Structural Bioinformatics entry 2HYI) is shown in light-blue sticks. (B) Inside view of the RNA binding area of the two RecA domains with the 5′ end of the binding site obstructed by the 5′HP and the 3′ end bounded by the RecA-1 and ratchet domains (Helix 1 and Helix 2 of the ratchet domain are indicated). (C) SDS–PAGE analysis of streptavidin agarose pulldown using a U₃₀ biotinylated RNA. All reactions contained Prp43p and bovine serum albumin (BSA) to reduce background binding and the indicated nucleotide or U₃₀-biotin. ‘M' denotes marker. CTD, carboxy-terminal domain; NTD, amino-terminal domain; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; ssRNA, single-stranded RNA; WHD, winged-helix domain.