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. 2019 May 7;47(8):4349-4362.
doi: 10.1093/nar/gkz150.

Structural Basis for RNA Translocation by DEAH-box ATPases

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Free PMC article

Structural Basis for RNA Translocation by DEAH-box ATPases

Florian Hamann et al. Nucleic Acids Res. .
Free PMC article

Abstract

DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3'-5' direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.

Figures

Figure 1.
Figure 1.
Structural overview of Prp22 from Chaetomium thermophilum and functional comparison of ctPrp22 and ctPrp43. (A) Structural overview of Prp22 from C. thermophilum. The model of ctPrp22 is displayed as a cartoon representation. Residues from the truncated N-terminus (549–556) are shown in black, the RecA1 domain (557–733) in orange, the RecA2 domain (734–909) in blue, the WH domain (WH; 910–977) in gray, the HB domain (HB; 978–1091) in wheat and the OB fold (OB; 1092–1175) in green. (B) ATPase assays show that ctPrp22 has an increased ATPase activity compared to ctPrp43, but both are stimulated by the presence of an A20-RNA. (C) ctPrp22 and ctPrp43 show the same binding mode toward an A20-RNA in dependence of an adenosine nucleotide. Both bind the ssRNA best in presence of AMPPNP and show less binding in absence of any adenosine nucleotide. The affinity toward ssRNA is drastically decreased when ADP is present. ATPase activity as well as RNA-binding experiments were determined in triplicates and error bars for each measured data point are depicted. The error of fit is indicated in the tables as +/-.
Figure 2.
Figure 2.
RNA interactions of ctPrp22. (A) The ssRNA binds in the RNA-binding tunnel between helicase core and C-terminal domains. The RNA and amino acids either hydrogen-bonding or stacking with nucleotides of the RNA are depicted as ball and stick models and colored according to the domain coloring of Figure 1. The numbering of residues exhibiting the conserved RNA-backbone interaction pattern with the 3′ region as seen as well in the ctPrp43+ADP-BeF3+RNA structure (PDB ID: 5lta) is highlighted as ovals, whereas residues displaying a unique interaction have a rectangular shape. (B) Overview of 5′ interactions leading to the unique conformation of this region. Bases of U1-U3 point in the opposite direction of the β-hairpin. Major contributors to this conformation are residues H1128, P1129 and F1134 of the OB-fold domain forming a stacking triad and R1012 from the HB domain (C).
Figure 3.
Figure 3.
Intrinsic mobility of RecA2 domain and comparison of open and closed conformations of the helicase core. (A) Both ctPrp22 molecules found in the asymmetric unit of the Apo structure are depicted as cartoon models. The RecA2 domains are colored in blue (Apo1) and red (Apo2), the RecA1 domains and C-terminal domains are depicted in gray. While the latter domains superpose virtually identically, the RecA2 domains show distinct positions. (B & C) All structures are depicted as semi-transparent cartoon models and colored according to Figure 1. Center of mass of the RecA-like domains are displayed as spheres and accordingly colored. In order to calculate the centers of mass for the same sets of atoms, the RecA1 and RecA2 domains of the ctPrp22-RNA complex were superimposed with the corresponding domains of the individual catalytic states and centers of mass for these superposed domains were determined. Adenosine nucleotide-bound DEAH-box ATPase structures adopt a closed helicase core conformation (B), whereas ADP/ATP-free structures are found in an open conformation (C).
Figure 4.
Figure 4.
DEAH-box ATPases translocate at a step-size of one RNA nucleotide per hydrolyzed ATP. The helicase cores of ctPrp43 and ctPrp22 with bound ssRNAs are depicted as cartoon models with a domain coloring according to Figure 1. (A) ATP-bound ctPrp43 exhibits a closed conformation of the helicase core with a stack of four RNA nucleotides bound to the RecA-like domains. (B) In the absence of an adenosine nucleotide the helicase core of ctPrp22 adopts an open conformation, which allows the accommodation of an additional RNA nucleotide in the binding tunnel leading to a bound five-nucleotide stack. In both conformations, the stack is interrupted by the β-hairpin of the RecA2 domain. (C) A continuous cycling of these states enables DEAH-box ATPases to translocate in 3′ to 5′ direction along a single-stranded RNA. Upon helicase core opening, an additional RNA nucleotide can be incorporated between the β-hairpin and the first RNA nucleotide of the four-nucleotide stack. The helicase core closure induced by the binding of ATP pushes the RNA through the binding tunnel.
Figure 5.
Figure 5.
A serine in motif V senses the catalytic state. (A) Motif V is located in the RecA2 domain in close proximity to the RNA-binding tunnel and the active site. (B) Motif V exhibits a helical conformation in the absence of adenosine nucleotides, as well as when ADP is bound. In the presence of ATP, the helix is disrupted and the motif adopts an alternative conformation. A serine in this motif senses the current catalytic state via different polar interactions with the bound ligands (C–E). In the adenosine nucleotide-free but RNA-bound state, the serine side chain hydrogen bonds with an ssRNA phosphate (C), and in the presence of ADP, the mainchain interacts with two water molecules coordinated by the active site magnesium (D). (E) When ATP is bound, the motif V helix is distorted by the interactions of the serine with numerous active site components. The mainchain interacts with the catalytic water molecule and a magnesium-coordinated water molecule. The side chain undergoes polar interactions with an active site glutamate and glutamine as well as with another water molecule coordinated by the magnesium. (F) The ATP-dependent interactions of the serine force motif V into an unfavored conformation. Upon ATP hydrolysis the motif is able to relax back into its helical conformation, which is translated into a repositioning of the RecA2 domain. (G) ATPase activity measurements of ctPrp22-S837A and ctPrp22-S837G mutants compared with wild-type ctPrp22 in absence and in presence of A20-ssRNA. The experiments were determined in triplicates and error bars for each measured data point are depicted. The error of fit is indicated in the table as +/-.

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