Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36 - PubMed (original) (raw)

Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36

Michael C Chen et al. Nature. 2018 Jun.

Abstract

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than most polymerases can exert1,2. Eukaryotic cells contain numerous helicases that can unfold G-quadruplexes 3 . The molecular basis of the recognition and unfolding of G-quadruplexes by helicases remains poorly understood. DHX36 (also known as RHAU and G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with extremely high affinity4-6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates 6 . DHX36 is a multi-functional helicase that has been implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, haematopoiesis, and embryogenesis in mice9-12. Here we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3' single-stranded DNA segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that, together with the OB-fold-like subdomain, selectively binds parallel G-quadruplexes. Comparison with unliganded and ATP-analogue-bound DHX36 structures, together with single-molecule fluorescence resonance energy transfer (FRET) analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core; by pulling on the single-stranded DNA tail, these rearrangements drive G-quadruplex unfolding one residue at a time.

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Figures

Extended Data Figure 1.. Sequence alignment of DHX36 orthologs.

The Bos taurus DHX36 construct used to solve the DHX36-DSM-DNA_Myc_ co-crystal structure (PDB ID: 5HVE), wild-type Bos taurus DHX36, Homo sapiens DHX36, Drosophila melanogaster DHX36,Herpegnathos saltator DHX36, Latrodectus hesperus DHX36, and the Chaetomium thermophilum Prp43 crystallization construct (PDB ID: 5D0U) are aligned with a 0.5 threshold for percent similarity (gray shading). The glycine-rich region is responsible for DHX36 recruitment to stress granules, but it is not necessary for DHX36 binding or resolution of G-quadruplexes. Identical residues are shaded in black. Secondary structure from the DHX36-DSM-DNA_Myc_ co-crystal structure is indicated above each alignment section, with arrow, rectangle and cone denoting α-helix, β-strand, and 310-helix, respectively. Secondary structure is color-coded by domain or subdomain as in Fig. 1. Alignment was performed with Clustal Omega (ref. 42) and depicted using BoxShade (

http://sourceforge.net/projects/boxshade/

Extended Data Figure 2.. Single-molecule FRET analysis of wild-type human DHX36 and bovine DHX36 constructs.

(a) Schematic of the smFRET assay,. See Extended Data Fig. 10 for FRET state assignments. (b) Binding of wild-type human DHX36 (ref. ; DHX36-WT) to the G-quadruplex substrate, induces a shift from a high to medium and low FRET states (gray and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type human DHX36 displays repetitive unfolding activity (ref. 22), as indicated by the oscillation between medium and low FRET states after binding to the G-quadruplex substrate (blue trace). (c) Binding of wild-type bovine DHX36 (incorporating a KKK192AAA mutation to prevent spontaneous proteolysis; DHX36-AAA) to the G-quadruplex substrate, induces a shift from a high to medium and low FRET states (gray and cyan histograms, respectively). The shift is interpreted as the binding of DHX36 to the G-quadruplex substrate. Upon buffer flow, dissociation is not observed (purple histogram). Wild-type bovine DHX36 (DHX36-AAA) displays repetitive unfolding activity, as indicated by the oscillation between low and medium FRET states after binding to the G-quadruplex substrate (blue trace). FRET traces are shown for two molecules. (d) Deletion of residues 111–159, mutation EEK435YYY, and mutation KDTK752AATA to generate DHX36-DSM does not impair G-quadruplex binding or repetitive unfolding activity. FRET traces are shown for two molecules. (e) Dwell time comparison between human DHX36-WT (gray bars), bovine wild-type DHX36 (DHX36-AAA, cyan bars) and bovine DHX36-DSM (orange bars). All three proteins show a comparable FRET range, and the two bovine constructs exhibit similar dwell times between the medium and low FRET states. Dwell times between the bovine constructs and the human construct are different, which likely arises from interspecies differences. Each experiment was performed three times. Data are reported as box dot plots, with the data center as the median ± standard error of 1000 dwell times from 200 representative molecules. Mutation of (f) motif IVa (“hook loop”), (g) the OB subdomain residue R856, and (h) OII does not result in impaired repetitive unfolding activity. However, partial dissociation following washing is observed with the (f) motif IVa and (h) OII mutation. (i) Pre-incubation of bovine DHX36-AAA with the non-hydrolyzable ATP γ-phosphate hydrolysis transition state mimic ADP•AlF4− does not affect repetitive unfolding activity on G-quadruplex substrates. (j) Addition of ATP (red arrow) while DHX36-AAA is displaying repetitive unfolding activity on G-quadruplex substrates results in DHX36 dissociation (blue arrow) on the seconds timescale. Each experiment was repeated three times producing highly similar results. Each measurement yields data from at least 10,000 molecules.

Extended Data Figure 3.. Electron density maps superimposed on refined structures.

(a) Portion of the density-modified 3.1 Å-resolution experimental SAD electron density map of selenomethionyl DHX36-core contoured at 1 s.d. above mean peak height, superimposed on a partially refined atomic model (Methods). (b) Portion of the 2.5 Å-resolution simulated-annealing omit 2|_F_o|-|_F_c| electron density map of DHX36-Core in complex with ADP•BeF3− (PDB ID: 5HVC) contoured at 1.5. (c) Portion of a simulated annealing-omit 2|_F_o|-|_F_c| electron density map of the DHX36-DSM-DNA_Myc_complex corresponding to the G-quadruplex, contoured at 1 s.d.. (d) Portion of the electron density map (c) corresponding to the OI loop and the DSM helix (lower left and right, respectively). A portion of the DNA is in the upper center.

Extended Data Figure 4.. Comparison of DNAMyc and DSM with solution structures of a _c_-Myc promoter sequence-derived parallel DNA G-quadruplex and DSM bound to a parallel DNA G-quadruplex.

(a) Cartoon representation of the _c-Myc_G-quadruplex structure, adopted by the DNA of sequence 5’ – TGA

GGG

TAA – 3 (PDB ID: 1XAV). (b) Schematic of the c-Myc G-quadruplex (PDB ID: 1XAV); compare with Fig. 2a. The DHX36-DSM-DNA_Myc_ co-crystal structure (PDB ID 5HVE; colored as in Fig. 1) was superimposed through the G-quadruplex with the solution structure (PDB ID: 2N21; gray) of a DSM-derived peptide bound to a G-quadruplex. (c) If the superposition is performed so that the 5' and 3' G-tracts of the G-quadruplexes from the two structures align, the α-helix of the solution structure of the DSM-derived peptide is oriented approximately 90° with respect to the DSM α-helix from the DHX36-DSM-DNA_Myc_co-crystal structure. (d) If arbitrarily rotated along the quadruplex 4-fold axis, the DSM α-helices from both structures approximately align. (e) Even with this rotation, the two structures differ in the DSM side chains presented to the DNA. (f),(g) Helical wheel representations of the DSM α-helices from the DHX36-DSM-DNA_Myc_co-crystal structure and the solution structure of the DSM-derived peptide bound to a G-quadruplex, respectively. Residues in cyan and bold make van der Waals contacts with the quadruplex face and hydrogen bond with the DNA backbone, respectively. Residue numbers correspond to the DHX36-DSM-DNA_Myc_ co-crystal structure.

Extended Data Figure 5.. Analysis of DNA_Myc_ conformers by differential scanning calorimetry (DSC).

(a) DNA constructs used in the analysis. DNA_Myc_, DNA used for co-crystallization with DHX36-DSM (Methods). Residues that form a three-tiered G-quadruplex and those that form propeller loops in the free DNA are boxed and underlined, respectively. 16-nt DNA_Myc_, DNA minimized to eliminate 5' and 3' single-stranded extensions to the G-quadruplex. 16-nt mutant DNA_Myc_, variant of the former with two mutations (red) to enforce the three quartets observed in the DHX36-DSM-DNA_Myc_ co-crystal structure. (b) Size-exclusion chromatograms (Methods) of 22-nt DNA_Myc_, 16-nt DNA_Myc_ and 16-nt mutant DNA_Myc_ in the presence of either 150 mM or 20 mM KCl, demonstrating greater conformational homogeneity of the DNAs at lower KCl concentration. (c) DSC thermograms (prior to buffer correction) for the three DNAs, in 20 mM KCl. Three independent experiments are plotted for each DNA. (d) Triplicate non-linear least-squares analyses of thermograms for the three DNAs. Black and red curves, buffer-corrected DSC data and curve-fits, respectively.T_m (melting temperature) and Δ_H (enthalpy change) are reported as means ± standard deviations. Each experiment was repeated three times with two sets of identical DNA preparations.

Extended Data Figure 6.. Alignments of the structures of DHX36, MLE, and Prp43

RecA1 domains were superimposed. Vectors from red to blue denote Cα displacement between identical or structurally homologous residues. (a) Superposition of DHX36-DSM-DNA_Myc_and unliganded DHX36-Core (5HVA) structures (green and orange, respectively). DNAMyc is pink. (b) Superposition of DHX36-DSM-DNA_Myc_ (green) and Prp43 (ref. 16) bound to rU16 and ADP•BeF3− (5LTA; blue; “ground”). DNA_Myc_ from the DHX36-DSM-DNA_Myc_ structure is in pink. (c) Superposition of Prp43 bound to rU8 and ADP•BeF3− (5LTA; blue; “ground”) to MLE (ref. 15) bound to rU15 and ADP•AlF4− (5AOR; silver; “transition”). DNA_Myc_ from the DHX36-DSM-DNA_Myc_ structure is in pink. (d) Superposition of MLE bound to rU15 and ADP•AlF4− (5AOR; silver; “transition”) and Prp43 bound, to ADP (3KX2/2XAU; gold; “post-hydrolysis). DNA_Myc_ from the DHX36-DSM-DNA_Myc_ structure is in pink. (e) Superposition of Prp43 bound to ADP (3KX2/2XAU; gold; “post-hydrolysis) to unliganded DHX36-Core (5HVA; magenta; “apo”).).

Extended Data Figure 7.. Model of the mechanochemical cycle of the DEAH/RHA helicase DHX36.

The domain motions are based on the superpositions in Extended Data Fig. 6. The orange, green, yellow, and blue blocks represent RecA1, RecA2, C-terminal domain, and N-terminal extension, respectively. The purple wedge represents OB. Bold dotted lines represent likely intrinsically disordered protein motifs that fold upon G-quadruplex binding. (a,b) In the absence of a G-quadruplex nucleic acid substrate, DHX36 cycles between an apo (or structurally indistinguishable ATP-bound) and post-hydrolysis states. (c,d) DHX36 binds the G-quadruplex substrate and pulls on it in the 3’-direction through concerted and opposite rotations of RecA2 and C-terminal domains. Oscillation of the RecA2 and C-terminal domains is likely responsible for the ATP-independent repetitive unfolding activity detected by smFRET (ref. , Extended Data Fig. 2 and Fig. 4). (d,e) Binding of ATP induces domain closure., (f,g) ATP hydrolysis yields a post-hydrolysis state that is incompatible with nucleic acid binding. ADP dissociates, and DHX36 is reset back to its apo state, (c). In addition to the rearrangement of motif Va (ref. 17), ATP hydrolysis is stimulated by nucleic acid binding likely because nucleic acid binding results in the opening of the helicase core. Diffusion into the NTP binding pocket is thus increased. Model (e) is based on the superposition in Extended Data Fig. 6b. Model (f) is based on the superposition in Extended Data Fig. 6c. Model (g) is based on the superposition in Extended Data Fig. 6d. Model (h) is based on the superposition in Extended Data Fig. 6e.).

Extended Data Figure 8.. Comparison of canonical and reorganized DNA_Myc_ G-quadruplex.

DNA_Myc_ǂ denotes the canonical DNA_Myc_ structure, whereas DNA_Myc_* represents the reorganized DNA_Myc_ found in the DHX36-DSM-DNA_Myc_ co-crystal structure. (a) Structure of the DNA_Myc_ǂ top G-quartet (PDB ID: 2N21). (b) Structure of the DNA_Myc_* top G-quartet. (c) Primary sequence alignment of the canonical and reorganized DNA_Myc_ G-quadruplex. Bold residues participate in formation of a quartet. (d) The structure of DNA_Myc_ G-quadruplex found in our co-crystal structure, represented here by DNA_Myc_*. Distances between Ade1-Thy24 as well as Gua16-Gua17, Gua17-Thy18, and Thy18-Thy19 are indicated. Theoretical FRET efficiencies (E) for DNA_Myc_ǂ and DNA_Myc_* were calculated using_E_ = 1/[1 + (r/R_0)6] where_R_0 = 53 Å for the Cy3-Cy5 pair and_r is the distance between Cy3-Cy5. Since smFRET experiments were performed with a DNA_Myc_G-quadruplex containing a 3' ssDNA extension of 9 thymines, we added the distance between two thymines to the theoretical FRET efficiency model assuming an average internucleotide distance of 7.1 Å. Since the difference between the hypothetical DNA_Myc_ǂ previously solved by NMR and DNA_Myc_* found in our co-crystal structure is 1 nucleotide, we modeled r_ǂ and_r* as 50.2 Å and 57.3 Å. From these parameters, we obtained predicted FRET efficiencies of 0.58 and 0.39 for DNA_Myc_ǂ and DNA_Myc_*, respectively. These predicted FRET efficiencies closely match the experimental oscillating FRET efficiencies of ~0.6 and ~0.4. (e) The high FRET state of ~0.85 is observed prior to DHX36 binding to the DNA_Myc_ G-quadruplex. (f) DHX36 initially binds to DNA_Myc_ǂ (~0.6). (g) Likely due to ATP-independent C-terminal domain rotations also observed16 with Prp43p, the DNA_Myc_, G-quadruplex is partially unwound to DNA_Myc_* (~0.4). DHX36 then oscillates between DNA_Myc_* and DNA_Myc_ǂ in an ATP-independent repetitive unfolding activity.

Figure 1. Overall structure of the DHX36 – G-quadruplex DNA complex.

(a) Domain organization; G-quadruplex (G4) and ssDNA-interacting regions indicated. (b) Cartoon representation of the co-crystal structure of DHX36 bound to DNAMyc, color-coded as ina. Spheres denote two disordered segments (blue, 20 and 53 residues in the crystallization constructand wild-type, respectively; and green 13 residues). OB loops I and II (OI and OII) contact DNA. (c) 90° rotation. (d) Electrostatic potential calculated omitting DNA from the co-crystal structure (blue to red, ±5_k_B_T_). (e) Phylogenetic conservation among 250 DHX36 orthologs (white to green, least to most conserved).

Figure 2. DHX36-DNA interaction.

(a) Schematic of the DHX36-bound all-parallel quadruplex. (b) The DHX36-specific motif (DSM) stacks on the 5’ (top) non-canonical quartet. Transparent spheres, van der Waals radii. (c) The DSM and the OI loop of OB flank Ade1. (d) Interaction of the DSM and loop OI of DHX36 with the DNA backbone near the 5’ of DNA_Myc_. (e) Interaction of the OB loop OII and the RecA2 domain with Thy18–Thy 22 of the 3’ single-stranded region of DNA_Myc_. (f) Interaction of the RecA1 domain and WH of DHX36 with Thy23–Thy24 of the 3’ single-stranded region of DNA_Myc_.

Figure 3. DNA-binding-induced structural transitions of DHX36.

(a) Superposition of DHX36-DSM-DNA_Myc_ and DHX36-Core structures (green and orange, respectively, Cα vectors from red to blue) through RecA1. DNA_Myc_, pink. (b) 90° view. Red circle and blue cross denote C-terminal domain rotation out of and into the plane, respectively. (c) C-terminal sub-domains unliganded and DNA-bound, pastel and solid colors, respectively. Black circle, approximate axis of rotation. (d) The WH in unliganded and DNA-bound states. Thy24 of DNA_Myc_ impinges on the loop linking WH and RL. (e) The OI and OII loops of OB in unliganded and DNA-bound states. (f) RecA2 in unliganded and DNA-bound states.

Figure 4. Single-molecule FRET analysis of DHX36-DSM mutants.

. (a) Reporter with a G-quadruplex of the DNA_Myc_ sequence, and a 9-thimidine single-stranded 3’ tail. DHX36 shifts FRET from high (~0.8) to medium–low oscillation (~0.6, canonical DNA_Myc_, ~0.4, reorganized DNA_Myc_, Extended Data Figs. 4,10). (b) Structure-guided mutations. (c) The DHX36-DSM crystallization construct remains DNA-bound upon flow and exhibits repetitive unfolding, similar to wild-type (Extended Data Fig. 2). (d) The Y69A mutant lacks repetitive unfolding and dissociates from G-quadruplex upon flow. (e)-(g) Three mutants remain bound following low, but lack repetitive unfolding. Each experiment was highly reproducible and in triplicate (data from >10,000 molecules/experiment).

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Structural basis of G-quadruplex unfolding by the DEAH/RHA helicase DHX36 - PubMed (original) (raw)