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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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The structure of yBrr2. (a) A schematic representation of the domain organization (abbreviations shown in parenthesis) of yBrr2. (b) Overall structure of the S2 domain, colored in rainbow spectrum from the N-terminus (blue) to the C-terminus (red). (c) Superimposition of the yeast (cyan) and human (yellow, PDB ID 2Q0Z) S2 domain structures. (d) Superimposition of the S2 domain and domains 4 and 5 of Hel308 (yellow, PDB ID 2P6R 23). The N-terminal helical domain, the middle helical domain, and the C-terminal Fn3 domain in S2 are colored blue, green, and purple, respectively. (e) A backbone model (by combining the Hel308 and S2 structure) depicting the overall structure of Brr2 consisting the NTD, Hel308-I, and Hel308-II. In each Hel308-like module, the D1-6 domains are colored in dark blue, light blue, green, purple, orange, and red, respectively. A partially unwound DNA duplex (black) as seen in the Hel308 structure was modeled in Hel308-I to represent the RNA substrate. The traditional H2 and S2 domain were indicated in ovals on the structural model.
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Figure 1: The structure of yBrr2. (a) A schematic representation of the domain organization (abbreviations shown in parenthesis) of yBrr2. (b) Overall structure of the S2 domain, colored in rainbow spectrum from the N-terminus (blue) to the C-terminus (red). (c) Superimposition of the yeast (cyan) and human (yellow, PDB ID 2Q0Z) S2 domain structures. (d) Superimposition of the S2 domain and domains 4 and 5 of Hel308 (yellow, PDB ID 2P6R 23). The N-terminal helical domain, the middle helical domain, and the C-terminal Fn3 domain in S2 are colored blue, green, and purple, respectively. (e) A backbone model (by combining the Hel308 and S2 structure) depicting the overall structure of Brr2 consisting the NTD, Hel308-I, and Hel308-II. In each Hel308-like module, the D1-6 domains are colored in dark blue, light blue, green, purple, orange, and red, respectively. A partially unwound DNA duplex (black) as seen in the Hel308 structure was modeled in Hel308-I to represent the RNA substrate. The traditional H2 and S2 domain were indicated in ovals on the structural model.

Mentions: To our knowledge, Brr2 is a the only DExD/H-box protein that contains two putative helicase domains, with the second helicase-like domain deviating more from the prototypical helicase motifs 14,19,20. Whereas the motifs in the first helicase-like domain of S. cerevisiae Brr2 (yBrr2) have been shown to be critical for ATPase activity, U4/U6 unwinding, and cell viability, the motifs in the second helicase domain can be disrupted with little consequence 12. Brr2 also contains multiple other domains, including an N-terminal domain and two Sec63 domains. The five domains are arranged as shown in Figure 1a and are abbreviated as N, H1, S1, H2, and S2. The Sec63 domain is defined by sequence similarity with Sec63 protein, a central component of the protein translocation apparatus on the ER membrane, although the structure and function of the Sec63 domain are unknown 21. With the exception of the H1 domain which is likely involved in RNA unwinding, the function of the other Brr2 domains is unclear.


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)

The structure of yBrr2. (a) A schematic representation of the domain organization (abbreviations shown in parenthesis) of yBrr2. (b) Overall structure of the S2 domain, colored in rainbow spectrum from the N-terminus (blue) to the C-terminus (red). (c) Superimposition of the yeast (cyan) and human (yellow, PDB ID 2Q0Z) S2 domain structures. (d) Superimposition of the S2 domain and domains 4 and 5 of Hel308 (yellow, PDB ID 2P6R 23). The N-terminal helical domain, the middle helical domain, and the C-terminal Fn3 domain in S2 are colored blue, green, and purple, respectively. (e) A backbone model (by combining the Hel308 and S2 structure) depicting the overall structure of Brr2 consisting the NTD, Hel308-I, and Hel308-II. In each Hel308-like module, the D1-6 domains are colored in dark blue, light blue, green, purple, orange, and red, respectively. A partially unwound DNA duplex (black) as seen in the Hel308 structure was modeled in Hel308-I to represent the RNA substrate. The traditional H2 and S2 domain were indicated in ovals on the structural model.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743687&req=5

Figure 1: The structure of yBrr2. (a) A schematic representation of the domain organization (abbreviations shown in parenthesis) of yBrr2. (b) Overall structure of the S2 domain, colored in rainbow spectrum from the N-terminus (blue) to the C-terminus (red). (c) Superimposition of the yeast (cyan) and human (yellow, PDB ID 2Q0Z) S2 domain structures. (d) Superimposition of the S2 domain and domains 4 and 5 of Hel308 (yellow, PDB ID 2P6R 23). The N-terminal helical domain, the middle helical domain, and the C-terminal Fn3 domain in S2 are colored blue, green, and purple, respectively. (e) A backbone model (by combining the Hel308 and S2 structure) depicting the overall structure of Brr2 consisting the NTD, Hel308-I, and Hel308-II. In each Hel308-like module, the D1-6 domains are colored in dark blue, light blue, green, purple, orange, and red, respectively. A partially unwound DNA duplex (black) as seen in the Hel308 structure was modeled in Hel308-I to represent the RNA substrate. The traditional H2 and S2 domain were indicated in ovals on the structural model.
Mentions: To our knowledge, Brr2 is a the only DExD/H-box protein that contains two putative helicase domains, with the second helicase-like domain deviating more from the prototypical helicase motifs 14,19,20. Whereas the motifs in the first helicase-like domain of S. cerevisiae Brr2 (yBrr2) have been shown to be critical for ATPase activity, U4/U6 unwinding, and cell viability, the motifs in the second helicase domain can be disrupted with little consequence 12. Brr2 also contains multiple other domains, including an N-terminal domain and two Sec63 domains. The five domains are arranged as shown in Figure 1a and are abbreviated as N, H1, S1, H2, and S2. The Sec63 domain is defined by sequence similarity with Sec63 protein, a central component of the protein translocation apparatus on the ER membrane, although the structure and function of the Sec63 domain are unknown 21. With the exception of the H1 domain which is likely involved in RNA unwinding, the function of the other Brr2 domains is unclear.

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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