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Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2.

Zhang L, Xu T, Maeder C, Bud LO, Shanks J, Nix J, Guthrie C, Pleiss JA, Zhao R - Nat. Struct. Mol. Biol. (2009)

Bottom Line: We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo.We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6.Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, Colorado, USA.

ABSTRACT
Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

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ATPase and helicase activities of Brr2 mutants. (a) Locations of residues corresponding to brr2-1 (E610G) and brr2-R1107A on the Hel308 structure (PDB ID 2P6R 23) and their relationship with the ssDNA (black). Color scheme is the same as 1e. (b) SDS PAGE of purified Brr2 WT, R1107A, and Brr2 S2del mutants. (c) Helicase assay of WT Brr2, Brr2 R1107A, and Brr2 S2del, in the absence and presence of Prp8-CTR. (d) ATPase activity of WT Brr2, Brr2 R1107A, and Brr2 S2del in the absence and presence of Prp8-CTR and U4/U6. (e) Quantification of the data in panel g. Error bars indicates s.e.m.
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Figure 2: ATPase and helicase activities of Brr2 mutants. (a) Locations of residues corresponding to brr2-1 (E610G) and brr2-R1107A on the Hel308 structure (PDB ID 2P6R 23) and their relationship with the ssDNA (black). Color scheme is the same as 1e. (b) SDS PAGE of purified Brr2 WT, R1107A, and Brr2 S2del mutants. (c) Helicase assay of WT Brr2, Brr2 R1107A, and Brr2 S2del, in the absence and presence of Prp8-CTR. (d) ATPase activity of WT Brr2, Brr2 R1107A, and Brr2 S2del in the absence and presence of Prp8-CTR and U4/U6. (e) Quantification of the data in panel g. Error bars indicates s.e.m.

Mentions: Two previously identified brr2 mutants are located in the Hel308-I module. Brr2-1 (E610G) and Brr2-R1107A are cs mutants that are defective in U4/U6 unwinding 11 and/or spliceosome disassembly 13. Residue E610 is located in motif Ib (TPEK in Brr2) of domain 1, which is typically involved in RNA or DNA substrate binding 23,30. The equivalent E123 residue of Hel308 is indeed on a helix right next to the ssDNA in the Hel308+DNA structure 23 (Fig. 2a). Interestingly, Hel308-W599 (the equivalent of Brr2-R1107, Supplementary Fig. 1) is on the midpoint of the ratchet helix of domain 4, across from motif 1b (Brr2-E610) on the opposite side of the same ssDNA (Fig. 2a). Hel308-W599 forms a stacking interaction with the 5th base of the ssDNA. Two helical-turns away, Hel308-R592 on the same helix interacts with the 3rd base of the ssDNA. In addition, the N-terminus of the ratchet helix interacts with motif IVa of domain 2.


Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2.

Zhang L, Xu T, Maeder C, Bud LO, Shanks J, Nix J, Guthrie C, Pleiss JA, Zhao R - Nat. Struct. Mol. Biol. (2009)

ATPase and helicase activities of Brr2 mutants. (a) Locations of residues corresponding to brr2-1 (E610G) and brr2-R1107A on the Hel308 structure (PDB ID 2P6R 23) and their relationship with the ssDNA (black). Color scheme is the same as 1e. (b) SDS PAGE of purified Brr2 WT, R1107A, and Brr2 S2del mutants. (c) Helicase assay of WT Brr2, Brr2 R1107A, and Brr2 S2del, in the absence and presence of Prp8-CTR. (d) ATPase activity of WT Brr2, Brr2 R1107A, and Brr2 S2del in the absence and presence of Prp8-CTR and U4/U6. (e) Quantification of the data in panel g. Error bars indicates s.e.m.
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Figure 2: ATPase and helicase activities of Brr2 mutants. (a) Locations of residues corresponding to brr2-1 (E610G) and brr2-R1107A on the Hel308 structure (PDB ID 2P6R 23) and their relationship with the ssDNA (black). Color scheme is the same as 1e. (b) SDS PAGE of purified Brr2 WT, R1107A, and Brr2 S2del mutants. (c) Helicase assay of WT Brr2, Brr2 R1107A, and Brr2 S2del, in the absence and presence of Prp8-CTR. (d) ATPase activity of WT Brr2, Brr2 R1107A, and Brr2 S2del in the absence and presence of Prp8-CTR and U4/U6. (e) Quantification of the data in panel g. Error bars indicates s.e.m.
Mentions: Two previously identified brr2 mutants are located in the Hel308-I module. Brr2-1 (E610G) and Brr2-R1107A are cs mutants that are defective in U4/U6 unwinding 11 and/or spliceosome disassembly 13. Residue E610 is located in motif Ib (TPEK in Brr2) of domain 1, which is typically involved in RNA or DNA substrate binding 23,30. The equivalent E123 residue of Hel308 is indeed on a helix right next to the ssDNA in the Hel308+DNA structure 23 (Fig. 2a). Interestingly, Hel308-W599 (the equivalent of Brr2-R1107, Supplementary Fig. 1) is on the midpoint of the ratchet helix of domain 4, across from motif 1b (Brr2-E610) on the opposite side of the same ssDNA (Fig. 2a). Hel308-W599 forms a stacking interaction with the 5th base of the ssDNA. Two helical-turns away, Hel308-R592 on the same helix interacts with the 3rd base of the ssDNA. In addition, the N-terminus of the ratchet helix interacts with motif IVa of domain 2.

Bottom Line: We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo.We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6.Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, Colorado, USA.

ABSTRACT
Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding during spliceosomal activation. Brr2 contains two helicase-like domains, each of which is followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. This, together with sequence similarities between Brr2's helicase-like domains and domains 1-3 of Hel308, led us to hypothesize that Brr2 contains two consecutive Hel308-like modules (Hel308-I and Hel308-II). Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism with Hel308. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2-Prp8-CTR complex to U4/U6. Our results have important implications for the mechanism and regulation of Brr2's activity in splicing.

Show MeSH
Related in: MedlinePlus