RNA Helicase Structure

1. RNA Helicase Structure Christie Carter Protein Structure and Function Fall 2006 Image: UAP56 helicase from helicase.net

2. Helicase Background • Use energy generated by NTP hydrolysis to break hydrogen bonds holding strands of DNA/RNA together. (DNA/RNA + Protein) • Eukaryotes, Prokaryotes, Viruses • Involved in repair, recombination, transcription, translation, etc. • There are 4 superfamilies (SF) of Helicases. • The RNA helicases belong to SF1 and SF2 (and SF3). • DNA helicases also belong to both families.

3. Superfamilies • The superfamilies are segregated based on motifs (not activity, substrate, source or direction of unwinding). • All have Walker A and B motifs in common. • Superfamilies 1 & 2 are the largest groups. These families contain most of the RNA Helicases. • Superfamily 3 contains helicases typically found in Viruses. This family contains mostly DNA helicases but does contain a few RNA helicases. • Superfamily 4 members come from bacterial systems. All DNA helicases. • Other families

4. Superfamilies 1 & 2 • Homologous in 7 motifs • Walker A (P-loop or Phosphate binding loop) • Ia. Oligonucleotide interaction motif • Walker B (ATP binding motif, DExx) • Unknown :“Sensor I” motif/helicase activity • Oligonucleotide interaction motif • Unknown/may participate in helicase activity • Arginine interacts with ATP Helicase Structure and Mechanism Cur Opin Struc Bio, 2002, 12:123-133

5. DEAD/H-box RNA Helicases • Superfamily 2 • Largest family of RNA helicases. • Share 9 conserved motifs including: Asp-Glu-Ala-Asp (DEAD/H): Walker B motif The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

6. Basic Structure of DEAD-Box Proteins Two covalently linked globular domains create a “Dumbell shape”

7. DEAD-box protein bound to ssRNA Q VI I III V II Ia IV Ib The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

8. Motifs • Many have poorly understood functions because of very few crystal structures. • So far, only 2 DEAD-box proteins have been cocrystallized with a bound nucleotide. • NO DEAD-box proteins have been cocrystallized with an RNA substrate. • Structural interactions and other information is based on information obtained with other (non-DEAD-box helicases).

9. Q-motif • An additional B-strand and 2 a-helices upstream of motif I • Forms a “cap” on top of domain I. • Potential ATP binding motif. The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

10. Motif I • AxTGoGKT • Walker A motif (P-loop, phosphate binding). • Common to all helicases. • Required for ATPase and helicase activity. • In crystal structures, this loop was shown to have 2 conformations (OPEN and CLOSED). • Open in nucleotide bound forms, closed in absence of nucleotide ligand. • In the closed state, ATP cannot be bound because of steric hindrance.

11. Motif I CLOSED OPEN The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

12. Motifs Ia and Ib • Ia – PTRELA, Ib – TPGR • Poorly studied. • May be necessary for RNA binding and structural rearrangements that occur through ATP binding. • Alanine substitutions in Ia of DEAD-box proteins alter ATPase and helicase activities; however, minor effect seen in DEAH-box proteins. • Structurally similar to IV and V, proposed to have similar function but not yet demonstrated The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

13. GG Doublet • Present in some but not all DEAD-box proteins. • Found in a loop between Ia and Ib. • Facilitate formation of a sharp turn. • Mutations in yeast eIF4a helicase affect growth. The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37 eIF4A MjDEAD

14. Motif II • Walker B motif – DEAD (Also DExD/H) • ATP binding domain. • Mutations in this motif alter ATPase and helicase activity, but do not affect RNA binding.

15. Motif III • SAT • Mutations in this motif lead to loss of helicase activity, while only minor effects on RNA binding, ATP binding/hydrolysis are seen. • SF1 motif III was shown to bind phosphates of ATP to serve to transduce information to motifs IV and V. • No evidence has shown this to be the case in DEAD-box proteins, although, it is thought that the function of motif III in DEAD-box proteins could be simlilar. • Possibly shifts into the inter-domain cleft during ATP binding

16. Motif III CLOSED OPEN Receptive to ATP

17. Motif IV • Minimal sequence: (L/V)IF • Poorly studied in DEAD-box proteins. • Potential RNA binding motif.

18. Motif V • TDVuARGID • RNA binding motif. • May play role in ATPase activity or helicase activity. • This motif interacts with motif VI. • Mutations in this motif affect growth.

19. Motif VI • HRIGRTGR • ATPase activity and RNA binding. • May interact with motif III. • The 2nd R may function as an “asparagine finger” which stabilizes water-Mg2+-phosphate intermediate in ATP hydrolysis.

20. DEAD-box protein bound to ssRNA Q VI I III V II Ia IV Ib The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

21. DEAD-box protein bound to ssRNA with ADP + Mg2+ present ADP decreases affinity for RNA substrate Q VI I III V II Ia IV Ib The DEAD-box protein family of RNA helicases, Cordin, Gene 2006, 367:17-37

22. Cool Websites and References • www.helicase.net • Cordin, O., et. al. 2006. The DEAD-box protein family of RNA helicases. Gene 367:17-37 • Linder, P, et. al. 2006. Bent out of shape: RNA unwinding by the DEAD-box helicase Vasa. Cell 125:219-221 • Sengoku, T., et. al. 2006. Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa. Cell 125:287-300 • Caruthers, J., et. al. 2002. Helicase structure and mechanism. Current Opinion in Structural Biology 12:123-133

23. Structural Basis for RNA Unwinding…Cell 2006, 125:287-300

24. Structural Similarity Structural Basis for RNA Unwinding…Cell 2006, 125:287-300