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  • Felisberto-Rodrigues, C.; Thomas, J.C.; McAndrew, C.; Le Bihan, Y.V.; Burke, R.; Workman, P.; van Montfort, R.L.M.
    Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release (2019), Biochem. J., 476, 2521-2543 .
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.6.4.13 ssRNA DHX8 ATPase activity is stimulated by RNA Homo sapiens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.6.4.13 recombinant expression of C-terminally His6-GST-tagged full-length DHX8 (fl-DHX8) and truncated construct containing the complete catalytically active C-terminal helicase core region (DHX8DELTA547) in Spodoptera frugiperda Sf9 cells using the baculovirus expression system Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.6.4.13 recombinant catalytic core fragment DHX8DELTA547 bound to ADP and to poly(A)6 single-strand RNA, hanging-drop vapour-diffusion, mixing of 500 nl of protein solution consisting of 3 mg/ml DHX8DELTA547, 1 mM ADP, and 1 mM MgCl2, with 0.00015 ml of reservoir solution comprising 15% v/v 2-methyl-2,4-pentanediol, 100 mM sodium acetate, pH 5.5 and 6% v/v DMSO, and equilibration over 0.25 ml reservoir solution. For the DHX8DELTA547 structure bound to poly(A)6 RNA, a DHX8DELTA547 crystal grown in the presence of ADP is soaked with 1 mM poly(A)6 ssRNA at 4°C for 48 h, X-ray diffraction structure determination and analysis at 2.3-3.2 A resolution, molecular replacement using the structures of RecA1 and RecA2 domains of Saccharomyces cerevisiae Prp43 (PDB ID 2XAU) and of helicase-associated domain of DHX8 (PDB ID 3I4U) as two independent search models Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
3.6.4.13 additional information isolation of the catalytically active helicase core DHX8DELTA547 enzyme fragment. The structure of DHX8DELTA547 reveals flexibility in the DEAH motif. The two DHX8DELTA547-ADP structures reveal a domain organisation very similar to that of other DEAH-box helicases. DHX8DELTA547 adopts a pyramidal-like structure with the two N-terminal RecA domains and the C-terminal ratchet-like and OB-fold domains on opposite sides of the putative DHX8 RNA-binding tunnel, and the C- and N-terminal domains connected by the WH domain. Comparison of kinetics of wild-type full-length enzyme and truncated mutant, overview Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.6.4.13 ADP the affinity for RNA is over 90fold weaker in the absence of nucleotide and in the presence of ADP, the RNA affinity is too weak to determine. These results indicate that DHX8-mediated disruption of RNA interactions occurs through a series of alternating strong and weak RNA binding events controlled by ATP hydrolysis. Both full-length fl-DHX8 and truncated DHX8DELAT547 preferentially bind the purine nucleotides ADP and GDP, but are also able to bind CDP, TDP and UDP. RNA binding triggers DEAH and P-loop movement and stimulates ADP release Homo sapiens
3.6.4.13 CDP
-
Homo sapiens
3.6.4.13 GDP
-
Homo sapiens
3.6.4.13 poly(A)10
-
Homo sapiens
3.6.4.13 poly(A)4
-
Homo sapiens
3.6.4.13 poly(A)6
-
Homo sapiens
3.6.4.13 poly(A)8
-
Homo sapiens
3.6.4.13 poly(C)10
-
Homo sapiens
3.6.4.13 poly(G)10
-
Homo sapiens
3.6.4.13 poly(U)10
-
Homo sapiens
3.6.4.13 poly(X) RNA DHX8 is an RNA-specific helicase by showing that a poly(dA)10 DNA strand cannot displace Cy5-poly(A)10 from DHX8 in the presence of ADP-AlFx. The displacement of the probe with poly(A)10, poly(C)10, poly(G)10 and poly(U)10 RNA indicates that DHX8 has a preference for binding adenine-rich sequences as the rank ascending order of IC50 values is poly (A)10, poly(U)10, poly(C)10, poly(G)10 Homo sapiens
3.6.4.13 TDP
-
Homo sapiens
3.6.4.13 UDP
-
Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.6.4.13 additional information
-
additional information Michaelis-Menten kinetics Homo sapiens
3.6.4.13 0.039
-
ATP pH 7.4, temperature not specified in the publication, recombinant wild-type full-length enzyme DHX8 Homo sapiens
3.6.4.13 0.04
-
ATP pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens
3.6.4.13 0.059
-
ATP pH 7.4, temperature not specified in the publication, recombinant wild-type full-length enzyme DHX8, in presence of poly(A)10 ssRNA Homo sapiens
3.6.4.13 0.121
-
ATP pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54, in presence of poly(A)10 ssRNA Homo sapiens

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.6.4.13 Mg2+ required Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.6.4.13 ATP + H2O Homo sapiens
-
ADP + phosphate
-
?

Organism

EC Number Organism UniProt Comment Textmining
3.6.4.13 Homo sapiens Q14562
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.6.4.13 recombinant His6-GST-tagged full-length DHX8 and truncated mutant DHX8DELTA547 from Sf9 insect cells by nickel affinity and heparin affinity chromatography, and gel filtration Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.4.13 ATP + H2O
-
Homo sapiens ADP + phosphate
-
?
3.6.4.13 ATP + H2O importance of the beta-phosphate for nucleotide binding Homo sapiens ADP + phosphate
-
?
3.6.4.13 additional information DHX8 has an in vitro binding preference for adenine-rich RNA, and RNA binding triggers the release of ADP through significant conformational flexibility in the conserved DEAH-, P-loop and hook-turn motifs. DHX8 makes base-specific contacts with RNA and preferentially binds adenine-rich RNA in vitro, RNA-bound DHX8DELTA547-A6 structure, overview. RNA binding triggers nucleotide release and establish the importance of R620 and both the hook-loop and hook-turn for DHX8 helicase activity, proposing that the hook-turn acts as a gatekeeper to aid correct directional RNA movement through the RNA-binding tunnel. Unwinding activity using an RNA/DNA duplex substrate comprising a 60-mer RNA strand (RNA60) annealed to a 30-mer DNA strand fluorescently labelled on the 30 end (DNA30-ATTO680). DHX8 is an RNA-specific helicase by showing that a poly(dA)10 DNA strand cannot displace Cy5-poly(A)10 from DHX8 in the presence of ADP-AlFx. The displacement of the probe with poly(A)10, poly(C)10, poly(G)10 and poly(U)10 RNA indicates that DHX8 has a preference for binding adenine-rich sequences as the rank ascending order of IC50 values is poly (A)10, poly(U)10, poly(C)10, and poly(G)10. Molecular details of RNA binding to DHX8, the RNA base stack is bookended by the beta-hairpin and the hook-turn hairpin, DHX8 forms RNA base-specific interactions through its OB-fold and RecA1 domains, overview Homo sapiens ?
-
-

Synonyms

EC Number Synonyms Comment Organism
3.6.4.13 DEAH-box RNA helicase
-
Homo sapiens
3.6.4.13 DEAH/RHA RNA helicase
-
Homo sapiens
3.6.4.13 DHX8
-
Homo sapiens
3.6.4.13 RNA helicase DHX8
-
Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.6.4.13 0.0283
-
ATP pH 7.4, temperature not specified in the publication, recombinant wild-type full-length enzyme DHX8 Homo sapiens
3.6.4.13 0.043
-
ATP pH 7.4, temperature not specified in the publication, recombinant wild-type full-length enzyme DHX8, in presence of poly(A)10 ssRNA Homo sapiens
3.6.4.13 0.1083
-
ATP pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens
3.6.4.13 0.3
-
ATP pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54, in presence of poly(A)10 ssRNA Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.6.4.13 7.4
-
assay at Homo sapiens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
3.6.4.13 additional information
-
additional information inhibition kinetics Homo sapiens

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
3.6.4.13 0.0026
-
pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens ADP
3.6.4.13 0.016
-
pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens poly(A)10
3.6.4.13 0.017
-
pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens poly(A)8
3.6.4.13 0.036
-
pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens poly(A)6
3.6.4.13 0.1
-
pH 7.4, temperature not specified in the publication, recombinant mutant enzyme DHX8DELTA54 Homo sapiens poly(A)4

General Information

EC Number General Information Comment Organism
3.6.4.13 evolution overall RNA binding mode is conserved between DHX8 and related SF2 helicases Homo sapiens
3.6.4.13 malfunction the affinity for RNA is over 90fold weaker in the absence of nucleotide and in the presence of ADP, the RNA affinity is too weak to determine. These results indicate that DHX8-mediated disruption of RNA interactions occurs through a series of alternating strong and weak RNA binding events controlled by ATP hydrolysis. Both full-length fl-DHX8 and truncated DHX8DELAT547 preferentially bind the purine nucleotides ADP and GDP, but are also able to bind CDP, TDP and UDP. DHX8 is an RNA-specific helicase by showing that a poly(dA)10 DNA strand cannot displace Cy5-poly(A)10 from DHX8 in the presence of ADP-AlFx. The displacement of the probe with poly(A)10, poly(C)10, poly(G)10 and poly(U)10 RNA indicates that DHX8 has a preference for binding adenine-rich sequences as the rank ascending order of IC50 values is poly (A)10, poly(U)10, poly(C)10, poly(G)10 Homo sapiens
3.6.4.13 additional information DHX8 is composed of a highly variable N-terminal domain, and a conserved C-terminal helicase domain. The latter contains two RecA domains, RecA1 and RecA2, that form the helicase core and contain up to 12 characteristic motifs that participate in ATP binding and hydrolysis, RNA binding and helicase activity. In addition to the RecA domains, the DHX8 helicase domain contains C-terminally located winged-helix (WH), ratchet-like and oligonucleotide binding (OB)-fold domains. RNA-bound DHX8DELTA547-A6 structure represents a state in the RNA translocation mechanism immediately following ADP release and shows extensive flexibility in the conserved DEAH motif and P-loop, which may facilitate nucleotide release. Compared with other helicase structures, DHX8DELTA547-A6 reveals unexpected differences in the interactions between the RNA substrate and the DEAH-specific hook-turn motif Homo sapiens
3.6.4.13 physiological function splicing is catalysed by the spliceosome, a large and dynamic protein-RNA complex consisting of five small nuclear ribonucleoproteins (snRNPs) and, in humans, about 200 accessory proteins. DHX8 is a crucial DEAH-box RNA helicase involved in splicing and required for the release of mature mRNA from the spliceosome. The DEAH/RHA RNA helicase DHX8 is required for the release of mature mRNA from the spliceosome Homo sapiens