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Crystal structure of the eukaryotic origin recognition complex.

Bleichert F, Botchan MR, Berger JM - Nature (2015)

Bottom Line: These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex.Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel.Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

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The Orc3 domain insertion forms a conserved, hydrophobic binding platform for Orc6. a) Surface representation of ORC. The Orc3 insertion, which extends from the Orc3 AAA+ lid subdomain and interacts with the C-terminal helix of Orc6, is boxed. b) Secondary structure representation of the boxed region shown in (a). The Orc3 insertion forms a bi-lobed, α-helical fold, three helices of which create a binding site for Orc6. c) Surface conservation of the Orc3 insertion. Conserved Orc3 residues cluster in the region that interacts with the Orc3 lid and in the Orc6 binding pocket. The latter region contacts highly conserved residues in Orc6 (Y225 and W228). d) Close-up view of Orc3•Orc6 interactions, showing a primarily hydrophobic binding site in Orc3 for Orc6 residues (Y225, W228, M232, A236). The Meier-Gorlin syndrome equivalent in Drosophila Orc6, Y225, appears positioned within hydrogen-bonding distance of E354 in Orc3 (dashed line). Colors are as in (b). e to h) Biochemical validation of the binding register for Drosophila Orc6. e) Close-up of the Orc6•Orc3 interface. Orc6-Ala236 faces a hydrophobic surface formed by Orc3 residues and is also in close proximity to a natural cysteine in Orc3 (Cys372). To validate the register of the short C-terminal Orc6 helix and the Orc6•Orc3 interface, we mutated Orc6-Ala236 to either glutamate, which we hypothesized would impede binding to ORC1-5 due to clashes with hydrophobic residues in Orc3, or to cysteine, which we presumed would not affect Orc3 binding but would allow site-specific crosslinking to Orc3-Cys372. f) Orc6A236E has a reduced affinity for the ORC1-5 complex. The C-terminal domains (CTDs) of wild-type (WT) Orc6, Orc6A236E or the Meier-Gorlin syndrome equivalent Orc6Y225S were each N-terminally labeled with Alexa Fluor 488 and tested for ORC1-5 binding using fluorescence anisotropy. As previously shown14, the C-terminal domain of Orc6 binds ORC1-5 with low nanomolar affinity, whereas the Y225S mutation strongly reduces binding. As predicted based on the structure of the Orc6•Orc3 interface, the A236E mutation also reduces the affinity of the Orc6-CTD for ORC1-5. Mean and standard deviations from three (for Orc6Y225S and Orc6A236E) or six (for wild-type Orc6) independent experiments are shown. g) Orc6A236C is able to bind to the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C were incubated with ORC1-5 (containing MBP-tagged Orc4) and subjected to pull-down experiments using amylose resin. Both Orc6-CTDWT and Orc6-CTDA236C co-purified with ORC1-5. The pull-down experiment was performed under non-reducing experimental conditions similar to the crosslinking experiment in panel (h). Asterisks mark two likely proteolytic fragments of Orc3. h) The Orc6-CTDA236C mutant, but not the wild-type Orc6-CTD, specifically crosslinks to Orc3 within the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C, either alone or in the presence of ORC1-5, was incubated with a bifunctional maleimide crosslinker and the proteins subsequently analyzed by SDS-PAGE. In reactions containing ORC1-5 and Orc6-CTDA236C, crosslinking gives rise to a novel band with higher molecular weight than Orc3; the appearance of this band correlates with a decrease in the amount of uncrosslinked Orc3 and Orc6-CTD, and does not appear with reactions containing ORC1-5 and wild-type Orc6-CTD, indicating that this species corresponds to an Orc3-Orc6 crosslink (a moderately strong higher molecular-weight band that appears in the absence of Orc6 likely corresponds to homotypic adducts between exposed cysteines in Orc3). These results are consistent with the structure, which places Orc6-Ala236 in close proximity to Orc3-Cys372. Note that ORC1-5 contained MBP-tagged Orc4 in (g) but that the tag was removed in (h).
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Figure 10: The Orc3 domain insertion forms a conserved, hydrophobic binding platform for Orc6. a) Surface representation of ORC. The Orc3 insertion, which extends from the Orc3 AAA+ lid subdomain and interacts with the C-terminal helix of Orc6, is boxed. b) Secondary structure representation of the boxed region shown in (a). The Orc3 insertion forms a bi-lobed, α-helical fold, three helices of which create a binding site for Orc6. c) Surface conservation of the Orc3 insertion. Conserved Orc3 residues cluster in the region that interacts with the Orc3 lid and in the Orc6 binding pocket. The latter region contacts highly conserved residues in Orc6 (Y225 and W228). d) Close-up view of Orc3•Orc6 interactions, showing a primarily hydrophobic binding site in Orc3 for Orc6 residues (Y225, W228, M232, A236). The Meier-Gorlin syndrome equivalent in Drosophila Orc6, Y225, appears positioned within hydrogen-bonding distance of E354 in Orc3 (dashed line). Colors are as in (b). e to h) Biochemical validation of the binding register for Drosophila Orc6. e) Close-up of the Orc6•Orc3 interface. Orc6-Ala236 faces a hydrophobic surface formed by Orc3 residues and is also in close proximity to a natural cysteine in Orc3 (Cys372). To validate the register of the short C-terminal Orc6 helix and the Orc6•Orc3 interface, we mutated Orc6-Ala236 to either glutamate, which we hypothesized would impede binding to ORC1-5 due to clashes with hydrophobic residues in Orc3, or to cysteine, which we presumed would not affect Orc3 binding but would allow site-specific crosslinking to Orc3-Cys372. f) Orc6A236E has a reduced affinity for the ORC1-5 complex. The C-terminal domains (CTDs) of wild-type (WT) Orc6, Orc6A236E or the Meier-Gorlin syndrome equivalent Orc6Y225S were each N-terminally labeled with Alexa Fluor 488 and tested for ORC1-5 binding using fluorescence anisotropy. As previously shown14, the C-terminal domain of Orc6 binds ORC1-5 with low nanomolar affinity, whereas the Y225S mutation strongly reduces binding. As predicted based on the structure of the Orc6•Orc3 interface, the A236E mutation also reduces the affinity of the Orc6-CTD for ORC1-5. Mean and standard deviations from three (for Orc6Y225S and Orc6A236E) or six (for wild-type Orc6) independent experiments are shown. g) Orc6A236C is able to bind to the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C were incubated with ORC1-5 (containing MBP-tagged Orc4) and subjected to pull-down experiments using amylose resin. Both Orc6-CTDWT and Orc6-CTDA236C co-purified with ORC1-5. The pull-down experiment was performed under non-reducing experimental conditions similar to the crosslinking experiment in panel (h). Asterisks mark two likely proteolytic fragments of Orc3. h) The Orc6-CTDA236C mutant, but not the wild-type Orc6-CTD, specifically crosslinks to Orc3 within the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C, either alone or in the presence of ORC1-5, was incubated with a bifunctional maleimide crosslinker and the proteins subsequently analyzed by SDS-PAGE. In reactions containing ORC1-5 and Orc6-CTDA236C, crosslinking gives rise to a novel band with higher molecular weight than Orc3; the appearance of this band correlates with a decrease in the amount of uncrosslinked Orc3 and Orc6-CTD, and does not appear with reactions containing ORC1-5 and wild-type Orc6-CTD, indicating that this species corresponds to an Orc3-Orc6 crosslink (a moderately strong higher molecular-weight band that appears in the absence of Orc6 likely corresponds to homotypic adducts between exposed cysteines in Orc3). These results are consistent with the structure, which places Orc6-Ala236 in close proximity to Orc3-Cys372. Note that ORC1-5 contained MBP-tagged Orc4 in (g) but that the tag was removed in (h).

Mentions: In the structure, Orc1-5 each comprises one AAA+ fold, followed by a single C-terminal WH domain (Extended Data Fig. 3). The AAA+ and WH regions co-associate, but are segregated between the two ring tiers (Fig. 1b, c and Supplementary Video 1). Interestingly, the collar of WH domains is rotationally offset from the AAA+ domains, leading to a domain-swapped organization wherein, apart from Orc2, the WH domain of one subunit packs against the AAA+ domain of its adjoining partner (Fig. 1c). Domain swapping is facilitated by long linkages between the AAA+ and WH modules, a region known to be conformationally flexible in archaeal Orc homologs (Extended Data Fig. 3)16–19. Other deviations from archetypal AAA+ structures include the absence of a small α-helical “lid” subdomain in the AAA+ fold of Orc2, a larger-than normal lid in Orc4, and a complete domain insertion in the lid of Orc3; the latter augmentation forms the protuberance that extends from the principal ORC body to bind Orc6 (Extended Data Figs. 3 and 4; Supplementary Discussion). Globally, the interdigitated interactions between the WH and AAA+ collars, as well as the extensive protein-protein contacts between adjoining WH domains indicate that the WH domains are an important determinant of ORC stability (5,660 Å2 of total surface area are buried between the WH domains, and 15,052 A2 between the WH and AAA+ tiers).


Crystal structure of the eukaryotic origin recognition complex.

Bleichert F, Botchan MR, Berger JM - Nature (2015)

The Orc3 domain insertion forms a conserved, hydrophobic binding platform for Orc6. a) Surface representation of ORC. The Orc3 insertion, which extends from the Orc3 AAA+ lid subdomain and interacts with the C-terminal helix of Orc6, is boxed. b) Secondary structure representation of the boxed region shown in (a). The Orc3 insertion forms a bi-lobed, α-helical fold, three helices of which create a binding site for Orc6. c) Surface conservation of the Orc3 insertion. Conserved Orc3 residues cluster in the region that interacts with the Orc3 lid and in the Orc6 binding pocket. The latter region contacts highly conserved residues in Orc6 (Y225 and W228). d) Close-up view of Orc3•Orc6 interactions, showing a primarily hydrophobic binding site in Orc3 for Orc6 residues (Y225, W228, M232, A236). The Meier-Gorlin syndrome equivalent in Drosophila Orc6, Y225, appears positioned within hydrogen-bonding distance of E354 in Orc3 (dashed line). Colors are as in (b). e to h) Biochemical validation of the binding register for Drosophila Orc6. e) Close-up of the Orc6•Orc3 interface. Orc6-Ala236 faces a hydrophobic surface formed by Orc3 residues and is also in close proximity to a natural cysteine in Orc3 (Cys372). To validate the register of the short C-terminal Orc6 helix and the Orc6•Orc3 interface, we mutated Orc6-Ala236 to either glutamate, which we hypothesized would impede binding to ORC1-5 due to clashes with hydrophobic residues in Orc3, or to cysteine, which we presumed would not affect Orc3 binding but would allow site-specific crosslinking to Orc3-Cys372. f) Orc6A236E has a reduced affinity for the ORC1-5 complex. The C-terminal domains (CTDs) of wild-type (WT) Orc6, Orc6A236E or the Meier-Gorlin syndrome equivalent Orc6Y225S were each N-terminally labeled with Alexa Fluor 488 and tested for ORC1-5 binding using fluorescence anisotropy. As previously shown14, the C-terminal domain of Orc6 binds ORC1-5 with low nanomolar affinity, whereas the Y225S mutation strongly reduces binding. As predicted based on the structure of the Orc6•Orc3 interface, the A236E mutation also reduces the affinity of the Orc6-CTD for ORC1-5. Mean and standard deviations from three (for Orc6Y225S and Orc6A236E) or six (for wild-type Orc6) independent experiments are shown. g) Orc6A236C is able to bind to the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C were incubated with ORC1-5 (containing MBP-tagged Orc4) and subjected to pull-down experiments using amylose resin. Both Orc6-CTDWT and Orc6-CTDA236C co-purified with ORC1-5. The pull-down experiment was performed under non-reducing experimental conditions similar to the crosslinking experiment in panel (h). Asterisks mark two likely proteolytic fragments of Orc3. h) The Orc6-CTDA236C mutant, but not the wild-type Orc6-CTD, specifically crosslinks to Orc3 within the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C, either alone or in the presence of ORC1-5, was incubated with a bifunctional maleimide crosslinker and the proteins subsequently analyzed by SDS-PAGE. In reactions containing ORC1-5 and Orc6-CTDA236C, crosslinking gives rise to a novel band with higher molecular weight than Orc3; the appearance of this band correlates with a decrease in the amount of uncrosslinked Orc3 and Orc6-CTD, and does not appear with reactions containing ORC1-5 and wild-type Orc6-CTD, indicating that this species corresponds to an Orc3-Orc6 crosslink (a moderately strong higher molecular-weight band that appears in the absence of Orc6 likely corresponds to homotypic adducts between exposed cysteines in Orc3). These results are consistent with the structure, which places Orc6-Ala236 in close proximity to Orc3-Cys372. Note that ORC1-5 contained MBP-tagged Orc4 in (g) but that the tag was removed in (h).
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Figure 10: The Orc3 domain insertion forms a conserved, hydrophobic binding platform for Orc6. a) Surface representation of ORC. The Orc3 insertion, which extends from the Orc3 AAA+ lid subdomain and interacts with the C-terminal helix of Orc6, is boxed. b) Secondary structure representation of the boxed region shown in (a). The Orc3 insertion forms a bi-lobed, α-helical fold, three helices of which create a binding site for Orc6. c) Surface conservation of the Orc3 insertion. Conserved Orc3 residues cluster in the region that interacts with the Orc3 lid and in the Orc6 binding pocket. The latter region contacts highly conserved residues in Orc6 (Y225 and W228). d) Close-up view of Orc3•Orc6 interactions, showing a primarily hydrophobic binding site in Orc3 for Orc6 residues (Y225, W228, M232, A236). The Meier-Gorlin syndrome equivalent in Drosophila Orc6, Y225, appears positioned within hydrogen-bonding distance of E354 in Orc3 (dashed line). Colors are as in (b). e to h) Biochemical validation of the binding register for Drosophila Orc6. e) Close-up of the Orc6•Orc3 interface. Orc6-Ala236 faces a hydrophobic surface formed by Orc3 residues and is also in close proximity to a natural cysteine in Orc3 (Cys372). To validate the register of the short C-terminal Orc6 helix and the Orc6•Orc3 interface, we mutated Orc6-Ala236 to either glutamate, which we hypothesized would impede binding to ORC1-5 due to clashes with hydrophobic residues in Orc3, or to cysteine, which we presumed would not affect Orc3 binding but would allow site-specific crosslinking to Orc3-Cys372. f) Orc6A236E has a reduced affinity for the ORC1-5 complex. The C-terminal domains (CTDs) of wild-type (WT) Orc6, Orc6A236E or the Meier-Gorlin syndrome equivalent Orc6Y225S were each N-terminally labeled with Alexa Fluor 488 and tested for ORC1-5 binding using fluorescence anisotropy. As previously shown14, the C-terminal domain of Orc6 binds ORC1-5 with low nanomolar affinity, whereas the Y225S mutation strongly reduces binding. As predicted based on the structure of the Orc6•Orc3 interface, the A236E mutation also reduces the affinity of the Orc6-CTD for ORC1-5. Mean and standard deviations from three (for Orc6Y225S and Orc6A236E) or six (for wild-type Orc6) independent experiments are shown. g) Orc6A236C is able to bind to the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C were incubated with ORC1-5 (containing MBP-tagged Orc4) and subjected to pull-down experiments using amylose resin. Both Orc6-CTDWT and Orc6-CTDA236C co-purified with ORC1-5. The pull-down experiment was performed under non-reducing experimental conditions similar to the crosslinking experiment in panel (h). Asterisks mark two likely proteolytic fragments of Orc3. h) The Orc6-CTDA236C mutant, but not the wild-type Orc6-CTD, specifically crosslinks to Orc3 within the ORC1-5 complex. Orc6-CTDWT or Orc6-CTDA236C, either alone or in the presence of ORC1-5, was incubated with a bifunctional maleimide crosslinker and the proteins subsequently analyzed by SDS-PAGE. In reactions containing ORC1-5 and Orc6-CTDA236C, crosslinking gives rise to a novel band with higher molecular weight than Orc3; the appearance of this band correlates with a decrease in the amount of uncrosslinked Orc3 and Orc6-CTD, and does not appear with reactions containing ORC1-5 and wild-type Orc6-CTD, indicating that this species corresponds to an Orc3-Orc6 crosslink (a moderately strong higher molecular-weight band that appears in the absence of Orc6 likely corresponds to homotypic adducts between exposed cysteines in Orc3). These results are consistent with the structure, which places Orc6-Ala236 in close proximity to Orc3-Cys372. Note that ORC1-5 contained MBP-tagged Orc4 in (g) but that the tag was removed in (h).
Mentions: In the structure, Orc1-5 each comprises one AAA+ fold, followed by a single C-terminal WH domain (Extended Data Fig. 3). The AAA+ and WH regions co-associate, but are segregated between the two ring tiers (Fig. 1b, c and Supplementary Video 1). Interestingly, the collar of WH domains is rotationally offset from the AAA+ domains, leading to a domain-swapped organization wherein, apart from Orc2, the WH domain of one subunit packs against the AAA+ domain of its adjoining partner (Fig. 1c). Domain swapping is facilitated by long linkages between the AAA+ and WH modules, a region known to be conformationally flexible in archaeal Orc homologs (Extended Data Fig. 3)16–19. Other deviations from archetypal AAA+ structures include the absence of a small α-helical “lid” subdomain in the AAA+ fold of Orc2, a larger-than normal lid in Orc4, and a complete domain insertion in the lid of Orc3; the latter augmentation forms the protuberance that extends from the principal ORC body to bind Orc6 (Extended Data Figs. 3 and 4; Supplementary Discussion). Globally, the interdigitated interactions between the WH and AAA+ collars, as well as the extensive protein-protein contacts between adjoining WH domains indicate that the WH domains are an important determinant of ORC stability (5,660 Å2 of total surface area are buried between the WH domains, and 15,052 A2 between the WH and AAA+ tiers).

Bottom Line: These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex.Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel.Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

Show MeSH
Related in: MedlinePlus