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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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Nucleotide binding by Orc1, Orc4 and Orc5. For panels (a) to (c), molecular replacement with the apo-ORC model was used to phase diffraction data collected from an ORC-ATPγS co-crystal. Positive Fo-Fc difference density contoured at different sigma levels reveals clear features for nucleotide binding to the AAA+ domains of: a) Orc1, b) Orc4, and c) Orc5. ATPγS is docked into the difference density for reference; due to the moderate (4.0 Å) resolution of the data, this structure was not refined. d) Modeling of canonical AAA+ interactions between Orc1 and Orc4, generated using the Orc4•Orc5 interaction as a reference. Upper panel: structural overview of modeled AAA+ domain positioning between Orc1 and Orc4. Lower panel: Close-up of the modeled Orc1•Orc4 ATPase site. Side chains (taken from their place in the apo-ORC model as a reference) are shown for conserved catalytically important residues. WA – Walker A, WB – Walker B, SI – Sensor I, SII – Sensor II, RF – arginine finger.
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Figure 13: Nucleotide binding by Orc1, Orc4 and Orc5. For panels (a) to (c), molecular replacement with the apo-ORC model was used to phase diffraction data collected from an ORC-ATPγS co-crystal. Positive Fo-Fc difference density contoured at different sigma levels reveals clear features for nucleotide binding to the AAA+ domains of: a) Orc1, b) Orc4, and c) Orc5. ATPγS is docked into the difference density for reference; due to the moderate (4.0 Å) resolution of the data, this structure was not refined. d) Modeling of canonical AAA+ interactions between Orc1 and Orc4, generated using the Orc4•Orc5 interaction as a reference. Upper panel: structural overview of modeled AAA+ domain positioning between Orc1 and Orc4. Lower panel: Close-up of the modeled Orc1•Orc4 ATPase site. Side chains (taken from their place in the apo-ORC model as a reference) are shown for conserved catalytically important residues. WA – Walker A, WB – Walker B, SI – Sensor I, SII – Sensor II, RF – arginine finger.

Mentions: A consequence of Orc1’s disposition within the complex is that its nucleotide binding cleft resides ~40 Å away from the arginine finger of Orc4. Importantly, comparison of the crystal structure with a prior 3D EM reconstruction of ATPγS-bound Drosophila ORC14 shows excellent agreement between the two models (Fig. 4c and Supplementary Video 2), indicating that the Orc1 conformation in the crystal corresponds to the predominant state of the complex in solution. Moreover, co-crystallization of ORC with the ATP analog ATPγS, while showing clear density for nucleotide binding to the Orc1, Orc4, and Orc5 AAA+ folds (Extended Data Fig. 7a–c), recapitulates the configuration seen in apo-ORC. Together, these data indicate that Drosophila Orc1 must undergo a large structural change to support ATPase activity, but that ATP-binding is itself insufficient to drive such a rearrangement in a majority of ORC particles.


Crystal structure of the eukaryotic origin recognition complex.

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

Nucleotide binding by Orc1, Orc4 and Orc5. For panels (a) to (c), molecular replacement with the apo-ORC model was used to phase diffraction data collected from an ORC-ATPγS co-crystal. Positive Fo-Fc difference density contoured at different sigma levels reveals clear features for nucleotide binding to the AAA+ domains of: a) Orc1, b) Orc4, and c) Orc5. ATPγS is docked into the difference density for reference; due to the moderate (4.0 Å) resolution of the data, this structure was not refined. d) Modeling of canonical AAA+ interactions between Orc1 and Orc4, generated using the Orc4•Orc5 interaction as a reference. Upper panel: structural overview of modeled AAA+ domain positioning between Orc1 and Orc4. Lower panel: Close-up of the modeled Orc1•Orc4 ATPase site. Side chains (taken from their place in the apo-ORC model as a reference) are shown for conserved catalytically important residues. WA – Walker A, WB – Walker B, SI – Sensor I, SII – Sensor II, RF – arginine finger.
© Copyright Policy - permissions-link
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368505&req=5

Figure 13: Nucleotide binding by Orc1, Orc4 and Orc5. For panels (a) to (c), molecular replacement with the apo-ORC model was used to phase diffraction data collected from an ORC-ATPγS co-crystal. Positive Fo-Fc difference density contoured at different sigma levels reveals clear features for nucleotide binding to the AAA+ domains of: a) Orc1, b) Orc4, and c) Orc5. ATPγS is docked into the difference density for reference; due to the moderate (4.0 Å) resolution of the data, this structure was not refined. d) Modeling of canonical AAA+ interactions between Orc1 and Orc4, generated using the Orc4•Orc5 interaction as a reference. Upper panel: structural overview of modeled AAA+ domain positioning between Orc1 and Orc4. Lower panel: Close-up of the modeled Orc1•Orc4 ATPase site. Side chains (taken from their place in the apo-ORC model as a reference) are shown for conserved catalytically important residues. WA – Walker A, WB – Walker B, SI – Sensor I, SII – Sensor II, RF – arginine finger.
Mentions: A consequence of Orc1’s disposition within the complex is that its nucleotide binding cleft resides ~40 Å away from the arginine finger of Orc4. Importantly, comparison of the crystal structure with a prior 3D EM reconstruction of ATPγS-bound Drosophila ORC14 shows excellent agreement between the two models (Fig. 4c and Supplementary Video 2), indicating that the Orc1 conformation in the crystal corresponds to the predominant state of the complex in solution. Moreover, co-crystallization of ORC with the ATP analog ATPγS, while showing clear density for nucleotide binding to the Orc1, Orc4, and Orc5 AAA+ folds (Extended Data Fig. 7a–c), recapitulates the configuration seen in apo-ORC. Together, these data indicate that Drosophila Orc1 must undergo a large structural change to support ATPase activity, but that ATP-binding is itself insufficient to drive such a rearrangement in a majority of ORC particles.

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