Structural insights into the mechanism of the DEAH-box RNA helicase Prp43
- Institute for Microbiology and Genetics, GZMB, Georg-August-University Göttingen, Germany
- Max Planck Institute for Biophysical Chemistry, Germany
Figures
Figure 1 with 4 supplements
Crystal structure of Prp43 in complex with U7-RNA and the ATP-analog ADP•BeF3-.
(a) Domain overview of ctPrp43. The bottom bar indicates the N-terminally truncated construct (ΔN, 61–764) used for crystallization. (b) Overall structure of ctPrp43ΔN•U7•ADP•BeF3-. Domains are …
Figure 1—figure supplement 1
Omit maps of a fraction of the U7-RNA from the ctPrp43ΔN•U7•ADP•BeF3- complex structure and of the active site of ctPrp43ΔN•ADP•BeF3-(HR) (Figure 2a).
(a) |Fo-Fc| omit map of nucleotides U3-U7 at 3σ level. The complete RNA molecule was omitted for map calculation. Coloring according to Figure 1a. (b) The ADP•BeF3-, the Mg2+ ion and five water …
Figure 1—figure supplement 2
The two alternative conformations of the U7-RNA in the ctPrp43ΔN•U7•ADP•BeF3- complex structure.
Coloring according to Figure 1a. Nucleotides U1-U3 are present in two alternative conformations (A and B) which exhibit after crystallographic refinement an occupancy of 54% and 46%, respectively. …
Figure 1—figure supplement 3
Overview of the B-factors of the U7-RNA in the ctPrp43ΔN•U7•ADP•BeF3- complex structure.
B-factors of the bound RNA range from 37 to 159 Ų. The B-factors of U1 to U3 are clearly elevated compared to U4 to U7.
Figure 1—figure supplement 4
Schematic representation of the NS3 HCV- and MLE-RNA interaction networks.
(a) Interactions between NS3 HCV•ADP•BeF3- (PDBid: 3o8r) and the bound U8-RNA are shown. Polar interactions are presented as dotted lines and stacking interactions as double lines. Residues which …
Figure 2 with 1 supplement
Structures of Prp43 with the bound ATP-analog ADP•BeF3- in different crystal forms.
(a) Overall structure of ctPrp43ΔN•ADP•BeF3- at high and (b) low resolution. ctPrp43ΔN•ADP•BeF3-(HR) crystalized in the orthorhombic space group P212121 and ctPrp43ΔN•ADP•BeF3-(LR) in the hexagonal …
Figure 2—figure supplement 1
The main chain of the Hook-Turn (RF motif) is present in two alternative conformations.
The A conformation (47%) is shown in orange and the B conformation (53%) in purple blue. In the A conformation a type I β -turn is formed and in the B conformation a type II β-turn.
Figure 3
Conformational changes between the ctPrp43ΔN•ADP complex structure (5d0u) (Tauchert et al., 2016), ctPrp43ΔN•U7•ADP•BeF3- (5lta), ctPrp43ΔN•ADP•BeF3-(HR) (5ltj) and ctPrp43ΔN•ADP•BeF3-(LR) (5ltk).
Structures were superposed via their RecA1 domains. In the top panel the back view is presented (rotated by 180° with respect to Figure 1b, Figure 2a and Figure 2b) and in the bottom panel the side …
Figure 4 with 3 supplements
Helicase activity and RNA-binding assays of ctPrp43 and mutants.
(a) The maximal unwinding velocity (nM/min) for a dsRNA with a 3’ overhang is shown. (b) RNA binding of 5’−6FAM-U16-RNA by ctPrp43 and ctPrp43-HT was determined via fluorescence anisotropy …
Figure 4—figure supplement 1
Position of the two introduced cysteine residues in the ctPrp43-IDSB mutant.
The Phe 181 in the RecA1 domain and the Asn 623 in the ratchet-like domain were substituted by cysteines. Owing to their close spatial proximity, a disulfide bridge can be formed which traps Prp43 …
Figure 4—figure supplement 2
ATPase activity of Prp43 and mutants (a) without further stimulation, (b) in the presence of a G-patch, (c) in the presence of a U16-RNA and (d) in the presence of a G-patch and a U16-RNA.
kcat and KM values are shown in Table 3. Error bars, s.d. (n = 3 independent measurements). For detailed information refer to the Materials and methods section.
Figure 4—figure supplement 3
Raw data of exemplary helicase activity measurements.
The typical curve progression of individual measurements for this fluorescence-based helicase assay is shown for ctPrp43+ctPfa1-GP (red), ctPrp43-IDSB+ctPfa1-GP (blue), ctPfa1-GP (green) and the …
Figure 5 with 1 supplement
Intron-lariat spliceosome (ILS) disassembly assays.
10–30% glycerol gradient sedimentation of purified yeast ILS (scILS) incubated in solution with ATP plus (a) no recombinant protein, (b) scPrp43 and cofactors scNtr(1•2), (c) ctPrp43 and scNtr(1•2), …
Figure 5—figure supplement 1
Isolation of intron-lariat spliceosomes (ILSs).
Activated spliceosomes (BactΔPrp2) assembled on Actin7 wild-type pre-mRNA in heat-inactivated splicing extracts from a prp2-1 yeast strain expressing a temperature-sensitive Prp2 mutant, were first …
Figure 6 with 1 supplement
Active site of Prp43 in the ATP- and ADP-bound state.
(a, Left panel) Active site of Prp43 with the bound ATP-analog ADP•BeF3- as present in ctPrp43ΔN•ADP•BeF3-(HR) (PDBid: 5ltj). The RecA1 domain is colored in light gray, the RecA2 domain in dark …
Figure 6—figure supplement 1
Superposition of the RecA-like domains of the ctPrp43ΔN•ADP•BeF3- (HR) structure with chain A of the MLE•U10•ADP•AlF4- complex (PDB 5aor) (Prabu et al., 2015).
The conformation of the helicase core is highly similar, also for all conserved helicase SF2 motifs, indicated by an r.m.s.d. value of 0.70 Å for 235 Cα for the superposition.
Figure 7 with 1 supplement
Position of the Hook-Turn and Hook-Loop in ctPrp43.
(a) The localization of the Hook-Turn in the RecA1 domain and of the Hook-Loop in the RecA2 domain in the ctPrp43ΔN•U7•ADP•BeF3- complex structure is shown. Domains are colored according to Figure 1a…
Figure 7—figure supplement 1
Partial sequence alignment of Prp43 from C. thermophilum to all DEAH-box RNA helicases from S. cerevisiae and of MLE from D. melanogaster to all yeast DExH-box RNA helicases.
The amino-acid sequence of ctPrp43 (G0RY84) was aligned to scPrp43 (P53131), scPrp2 (P20095), scPrp16 (P15938), scPrp22 (P24384), scDhr1 (Q04217) and scDhr2 (P36009). The sequence of dmMLE (P24785) …
Videos
Video 1
Prp43 adopts an open conformation after ATP binding and switches into the closed conformation after binding to RNA.
This morphing movie between the ADP-bound state of Prp43 (PDBid: 5d0u), the two ADP•BeF3- bound states (PDBids: 5ltk and 5ltj) and the ctPrp43ΔN•U7•ADP•BeF3- complex structure (PDBid: 5lta) …
Video 2
Local conformational rearrangements at the active site induce global conformational changes in Prp43 which are coupled with the unwinding activity.
The morphing between the ctPrp43ΔN•U7•ADP•BeF3- (PDBid: 5lta) complex structure and ctPrp43ΔN•ADP (PDBid: 5d0u) reveals conformational rearrangements in the course of ATP hydrolysis, mainly of the …
Tables
Table 1
Data collection and refinement statistics.
ctPrp43ΔN•U7•ADP•BeF3- | ctPrp43ΔN•ADP•BeF3-(HR) | ctPrp43ΔN•ADP•BeF3-(LR) | |
|---|---|---|---|
PDBid | 5lta | 5ltj | 5ltk |
Data collection | |||
Space group | P6122 | P212121 | P65 |
Cell dimensions | |||
a, b, c (Å) | 106.39, 106.39, 356.70 | 88.83, 105.64, 119.05 | 184.34, 184.34, 82.32 |
α, β, γ (°) | 90.0, 90.0, 120.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 120.0 |
Resolution (Å) | 48.56 – 2.62 (2.70 – 2.62) | 79.02 – 1.78 (1.89 – 1.78) | 92.17 – 3.24 (3.39 – 3.24) |
Rmeas (%) | 5.9 (90.3) | 7.2 (123.5) | 9.8 (76.2) |
I/σ(I) | 22.16 (1.85) | 14.88 (1.64) | 14.65 (2.26) |
CC1/2 (%) | 99.9 (65.2) | 99.9 (61.9) | 99.8 (63.8) |
Completeness (%) | 98.5 (86.8) | 99.6 (98.8) | 98.8 (94.9) |
Redundancy | 5.14 (5.34) | 4.71 (4.66) | 4.00 (3.59) |
Refinement | |||
Resolution (Å) | 48.56 – 2.62 | 67.99 – 1.78 | 92.17 – 3.24 |
No. reflections | 36887 | 107276 | 25304 |
Rwork / Rfree | 19.70/22.97 | 17.67/19.88 | 18.22/21.78 |
No. atoms | |||
Protein | 5605 | 5730 | 5622 |
RNA | 204 | / | / |
Ligand / Ion | 37 | 105 | 42 |
Water | 52 | 688 | 4 |
B-factors(Ų) | |||
Protein | 72.83 | 34.41 | 88.87 |
RNA | 88.65 | / | / |
Ligand / Ion | 55.04 | 47.83 | 83.89 |
Water | 60.51 | 44.29 | 62.19 |
R.m.s. deviations | |||
Bond length (Å) | 0.0028 | 0.0041 | 0.0026 |
Bond angles (°) | 0.78 | 0.87 | 0.67 |
Ramachandran Plot | |||
Favored | 95.98 | 97.34 | 96.28 |
Outlier | 0.0 | 0.0 | 0.0 |
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Values in parentheses are for the highest resolution shell.
Table 2
Helicase activity.
nM/min | ± | |
|---|---|---|
ctPrp43 | 0.54 | 0.18 |
ctPrp43 + ctPfa1-GP | 60.03 | 2.75 |
ctPrp43 + ctNtr1-GP | 1.98 | 0.45 |
ctPfa1-GP | 0.46 | 0.28 |
ctPrp43-IDSB + ctPfa1-GP | 12.18 | 0.23 |
ctPrp43-HT+ ctPfa1-GP | 0.68 | 0.16 |
ctPrp43-HL + ctPfa1-GP | 61.87 | 7.14 |
ctPrp43-HT&HL+ ctPfa1-GP | 0.88 | 0.03 |
Table 3
ATPase activity.
kcat (min−1) | ± | KM (µM) | ± | |
|---|---|---|---|---|
ctPrp43 | 1.81 | 0.03 | 47.23 | 3.40 |
ctPrp43 + U16-RNA | 2.08 | 0.05 | 3.75 | 0.68 |
ctPrp43 + ctNtr1-GP | 4.16 | 0.04 | 5.49 | 0.32 |
ctPrp43 + ctNtr1-GP + U16-RNA | 16.28 | 0.09 | 4.87 | 0.19 |
ctPrp43 + ctPfa1-GP | 36.28 | 0.36 | 8.90 | 0.58 |
ctPrp43 + ctPfa1-GP + U16-RNA | 372.20 | 2.91 | 26.11 | 1.21 |
ctPrp43-IDSB | 5.04 | 0.12 | 16.23 | 2.38 |
ctPrp43-IDSB + ctPfa1-GP | 199.35 | 1.59 | 29.04 | 1.35 |
ctPrp43-IDSB + U16-RNA | 24.99 | 0.37 | 3.42 | 0.38 |
ctPrp43-IDSB + ctPfa1-GP + U16-RNA | 609.38 | 6.15 | 88.14 | 3.50 |
ctPrp43-HT | 13.21 | 0.18 | 91.23 | 5.89 |
ctPrp43-HT + ctPfa1-GP | 60.95 | 0.74 | 13.29 | 1.03 |
ctPrp43-HT + U16-RNA | 10.58 | 0.34 | 53.92 | 8.27 |
ctPrp43-HT + ctPfa1-GP + U16-RNA | 98.61 | 1.84 | 13.19 | 1.52 |
ctPrp43-HL | 6.19 | 0.05 | 14.31 | 0.71 |
ctPrp43-HL + ctPfa1-GP | 86.50 | 0.84 | 14.38 | 0.89 |
ctPrp43-HL + U16-RNA | 6.16 | 0.06 | 5.38 | 0.35 |
ctPrp43-HL + ctPfa1-GP + U16-RNA | 533.55 | 9.53 | 49.49 | 3.86 |
ctPrp43-HT&HL | 7.61 | 0.11 | 16.72 | 1.49 |
ctPrp43-HT&HL + ctPfa1-GP | 113.18 | 1.03 | 25.17 | 1.37 |
ctPrp43-HT&HL + U16-RNA | 5.53 | 0.07 | 5.59 | 0.51 |
ctPrp43-HT&HL + ctPfa1-GP + U16-RNA | 82.28 | 1.31 | 11.88 | 1.18 |
