The structure of Prp2 bound to RNA and ADP-BeF3- reveals structural features important for RNA unwinding by DEAH-box ATPases

Abstract

Noncoding intron sequences present in precursor mRNAs need to be removed prior to translation, and they are excised via the spliceosome, a multimegadalton molecular machine composed of numerous protein and RNA components. The DEAH-box ATPase Prp2 plays a crucial role during pre-mRNA splicing as it ensures the catalytic activation of the spliceosome. Despite high structural similarity to other spliceosomal DEAH-box helicases, Prp2 does not seem to function as an RNA helicase, but rather as an RNA-dependent ribonucleoprotein particle-modifying ATPase. Recent crystal structures of the spliceosomal DEAH-box ATPases Prp43 and Prp22, as well as of the related RNA helicase MLE, in complex with RNA have contributed to a better understanding of how RNA binding and processivity might be achieved in this helicase family. In order to shed light onto the divergent manner of function of Prp2, an N-terminally truncated construct of Chaetomium thermophilum Prp2 was crystallized in the presence of ADP-BeF₃^- and a poly-U₁₂ RNA. The refined structure revealed a virtually identical conformation of the helicase core compared with the ADP-BeF₃^-- and RNA-bound structure of Prp43, and only a minor shift of the C-terminal domains. However, Prp2 and Prp43 differ in the hook-loop and a loop of the helix-bundle domain, which interacts with the hook-loop and evokes a different RNA conformation immediately after the 3' stack. On replacing these loop residues in Prp43 by the Prp2 sequence, the unwinding activity of Prp43 was abolished. Furthermore, a putative exit tunnel for the γ-phosphate after ATP hydrolysis could be identified in one of the Prp2 structures.

Keywords: DEAH-box ATPases; Prp2; Prp43; RNA helicases; spliceosome.

Figure 1

Structural overview of the pre-catalytic state of Prp2. (a) Prp2 and the bound RNA are displayed as a cartoon model and ADP-BeF₃ ⁻ is depicted as sticks. The crystallized construct is composed of two RecA-like domains (RecA1, orange; RecA2, blue), a winged-helix (WH) domain (gray), a helix-bundle (HB) domain (wheat) and a oligosaccharide-binding (OB) domain (green). The RNA is bound between the helicase core and the C-terminal domains and the nucleotide is sandwiched between the RecA-like domains. Conserved residues interacting with the RNA backbone of the 3′ stacked region are shown in circles, whereas the remaining interacting residues are shown in rectangular shapes. (b) Superposition of active-site residues of Prp2 and Prp43 interacting with ADP-BeF₃ ⁻ (pale green and blue), the magnesium ion (green) and coordinated water molecules (red). Both DEAH-box ATPases interact with the nucleotide in an identical manner. (c) Superposition of all structurally characterized DExH-box ATPases bound to an ATP analog. The conformation of the helicase core in the ATP-bound state is highly conserved.

Figure 2

Movements of conserved sequence motif III control the formation of a channel connecting the nucleotide-binding site to the protein surface. (a) In the ctPrp2–ADP-CF1 structure, motif III adopts a conformation that allows the formation of a channel that connects the γ-phosphate position of the active site to the exterior of the protein. In other ADP- and ADP-BeF₃ ⁻-bound structures motif III closes this channel. The exit channel is highlighted as purple spheres. CF stands for crystal form. (b) Overview of motif III interactions in the ADP-bound state. (c) Overview of motif III interactions in the ADP-BeF₃ ⁻-bound state. (d) Exemplary trajectory of the γ-phosphate through the exit channel based on MD calculations. (e) Only minor movements of motifs I and III allow trespassing of the γ-phosphate through the exit channel (green, ATP-bound state; red, moved motifs after MD calculations).

Figure 2

Movements of conserved sequence motif III control the formation of a channel connecting the nucleotide-binding site to the protein surface. (a) In the ctPrp2–ADP-CF1 structure, motif III adopts a conformation that allows the formation of a channel that connects the γ-phosphate position of the active site to the exterior of the protein. In other ADP- and ADP-BeF₃ ⁻-bound structures motif III closes this channel. The exit channel is highlighted as purple spheres. CF stands for crystal form. (b) Overview of motif III interactions in the ADP-bound state. (c) Overview of motif III interactions in the ADP-BeF₃ ⁻-bound state. (d) Exemplary trajectory of the γ-phosphate through the exit channel based on MD calculations. (e) Only minor movements of motifs I and III allow trespassing of the γ-phosphate through the exit channel (green, ATP-bound state; red, moved motifs after MD calculations).

Figure 3

Comparison of ssRNA binding to spliceosomal DEAH-box ATPases. All ssRNAs bound to spliceosomal DEAH-box ATPases exhibit a kink in the 5′ region. When the RecA2 domains are superimposed [Prp2/Prp43 in (a), Prp2/Prp22 in (b) and Prp43/Prp22 in (c)], the kinks in the Prp43 and Prp22 structures share a similar position and only the kink in the Prp2 structure is differently positioned.

Figure 4

The C-terminal loop dictates the conformation of the 5′ RNA region. (a) Superposition of Prp2 and Prp43 in the pre-catalytic state via the helicase core. The 3′ stacked RNA region superposes almost identically, but at the beginning of the 5′ region the RNA bound to Prp43 interacts with the C-terminal loop. This loop has a different conformation in Prp2 and does not interact with the RNA. (b) The base of the first nucleotide of the 5′ RNA region interacts with a proline and a serine of the Prp43 C-terminal loop. (c) The alternative conformation of the Prp2 C-terminal loop is stabilized by interactions with surrounding residues belonging to the helix-bundle domain and the hook-loop of the RecA2 domain.

Figure 5

Sequence conservation of the C-terminal loop and hook-loop. Sequence alignment of the C-terminal loop (a) and the hook-loop (b) of Prp2, Prp43, Prp16 and Prp22 in Chaetomium thermophilum, Saccharomyces cerevisiae, Homo sapiens, Xenopus laevis and Caenorhabditis elegans. (c) Helicase activities of various ctPrp43 constructs with a mutated C-terminal loop or/and hook-loop. An overview of the activities is shown as a bar plot and the k _obs values are listed below. All experiments were performed in triplicate; the standard deviation is highlighted as error bars and indicated as ± in the table.