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. 2015 Mar 19;519(7543):321-6.
doi: 10.1038/nature14239. Epub 2015 Mar 11.

Crystal structure of the eukaryotic origin recognition complex

Franziska Bleichert  1 , Michael R Botchan  2 , James M Berger  1
Affiliations

Crystal structure of the eukaryotic origin recognition complex

Franziska Bleichert et al. Nature. .

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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Figures

Extended Data Figure 1
Extended Data Figure 1
Deletion of variable N-terminal extensions in Orc1, Orc2 and Orc3 alters neither ORC stability nor overall ORC architecture. a) Gel-filtration chromatography trace of the ORC core used for crystallography, together with b) SDS-PAGE of respective ORC peak fractions from (a), indicate the formation of a stable hexameric complex. c) Full-length ORC and ORC containing N-terminal truncations display similar structural features in 2D EM class averages. Both complexes were imaged by negative-stain EM in the presence of ATPγS. Note that although class averages from ORC with truncated Orc1-3 subunits contain full-length Orc6, Orc6 is not visible due to its flexibile nature. Class averages for full-length ORC are derived from a dataset used in.
Extended Data Figure 2
Extended Data Figure 2
Experimental electron density contoured at 1σ for different regions of the ORC structure. The Orc1 winged-helix (WH) domain is shown in (a), the Orc3 insertion in (b), and the Orc4 AAA+ domain in (c).
Extended Data Figure 3
Extended Data Figure 3
Structure of individual ORC subunits compared to S. solfataricus Orc1-1 (PDB code 2qby chain A) and A. pernix Orc2 (PDB codes 1w5s chain A (left) and 1w5t chain C (right)). Different structural elements are colored as indicated. The initiator specific motif (ISM) of the AAA+ ATPase fold is shown in the inset. No electron density was observed for the region linking the AAA+ and WH domains of Orc5 (indicated by a dashed line). The very N-terminal region of Orc2, which could only be built as stretches of polyalanine, is not shown.
Extended Data Figure 4
Extended Data Figure 4
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 shown, 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).
Extended Data Figure 5
Extended Data Figure 5
ATP-binding site configuration at the Orc4•Orc5 and Orc5•Orc3 interfaces. a) Inter-AAA+ interactions between Orc4 and Orc5 are similar to canonical AAA+ interactions between DnaA protomers (top panel, only Orc4 is used for superpositioning onto the left (light gray) AAA+ domain of an ATP-bound DnaA dimer, PDB code 2hcb). Close-up views of the nucleotide-binding site are shown for Orc4 (bottom panel) and for DnaA for comparison (middle panel). The resemblance of the Orc4 nucleotide-binding pocket to the active site of functional AAA+ ATPases is somewhat surprising considering that mutations in the active site of Drosophila and human Orc4 have no reported effect on the ATPase activity of ORC as measured in vitro,, but may help explain why a Drosophila ORC mutant bearing a Walker A or B substitution in Orc4 exhibits modest DNA replication defects in extracts. b) The putative arginine finger in Orc5 is well conserved across homologs. A sequence logo of a multiple sequence alignment of the region containing the putative arginine finger (marked with an arrow) in eukaryotic Orc5 protein sequences is shown. Amino acid numberings correspond to the Drosophila Orc5 sequence. c) A potential Sensor-II equivalent arginine (marked with an arrow) in the Orc4 Walker A motif is conserved in eukaryotic Orc4 homologs. A sequence logo of the Walker A motif from a multiple sequence alignment of eukaryotic Orc4 protein sequences is shown. Amino acid positions are numbered as in Drosophila Orc4. d) Inter-AAA+ interactions between Orc5 and Orc3. The top panel shows a superposition derived from placing the AAA+ domain of Orc5 atop the AAA+ domain of the left (dark gray) protomer of an ATP-bound DnaA dimer; the bottom panel shows a close-up view of the nucleotide-binding site at the Orc5•Orc3 interface. Side chains of conserved residues known to be involved in nucleotide binding and hydrolysis in AAA+ ATPases are represented as sticks in both (a) and (d). WA – Walker A, WB – Walker B, SI – Sensor I, SII – Sensor II, RF – arginine finger.
Extended Data Figure 6
Extended Data Figure 6
The conformation of Orc1 arises from a reorientation between its AAA+ and WH domains, and not from changes within the AAA+ ATPase domain itself. a) Superpositioning of the WH domains of Orc1 and S. solfataricus Orc1-1 (PDB code 2qby chain A) reveals different conformations for both proteins, resulting from a large domain rotation of the Orc1 AAA+ domain around a pivot point in the linker preceding its WH domain. b) The Orc1 conformation is most similar to a state seen for A. pernix Orc2 (PDB code 1w5t chain C). The WH domains of both proteins were superposed as in (a). c to e) Superposing the AAA+ base subdomains of Orc1 and S. solfataricus Orc1-1 (panel (c), PDB code 2qby chain A), A. pernix Orc2 (panel (d), PDB code 1w5t chain C), and Orc3, Orc4 or Orc5 (panel (e)) shows that the typical AAA+ configuration between the base and lid subdomains are maintained in Orc1. Only a slight opening of the nucleotide-binding cleft is observed in Orc1, which is likely due to the absence of bound nucleotide. f and g) The most C-terminal α-helix of the Orc1 WH domain mediates interactions with the Orc1 lid subdomain. An overview of the interaction is shown in (f), with a close-up view of contacts between a conserved tyrosine (Tyr915) in the C-terminal Orc1 helix and a hydrophobic pocket of the Orc1 lid depicted in (g). h) The tyrosine in the C-terminal helix of Orc1 is well conserved across metazoan but not fungal Orc1 homologs. Alignments are shown as sequence logos. The numbering of amino acids is based on Drosophila Orc1, and the tyrosine is marked by an arrow.
Extended Data Figure 7
Extended Data Figure 7
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.
Extended Data Figure 8
Extended Data Figure 8
Docking of the observed and remodeled ORC structures into the cryo-EM density of S. cerevisiae and Drosophila ORC indicates that the ATPase domain of Orc1 is repositioned into a canonical AAA+/AAA+ interaction with Orc4 when Cdc6 is present, and supports a model where DNA passes through the central channel in ORC. a) The 3D EM volume for S. cerevisiae ORC (as present in a complex with Cdc6, Cdt1, and MCM2-7 and assembled in the presence of DNA – EMD-5625) contains Orc1 in the activated conformation. ORC with Orc1 in the autoinhibited conformation (left panel, as observed in the crystal structure) and remodeled conformation (right panel, remodeled) were docked into the ORC•Cdc6•Cdt1•Mcm2-7 cryo-EM map (only the density for ORC•Cdc6 is shown). The ORC•Cdc6 EM density readily accommodates Orc1 in the activated conformation, but not in its autoinhibited state. The EM density corresponding to Cdc6 is indicated in the right panel. b) DNA passes through the central ORC channel in the DNA•ORC•Cdc6 complex. ORC (with Orc1 in the remodeled conformation) was first docked into the cryo-EM map derived from a DNA•ORC•Cdc6 complex (EMD-5381). The AAA+ domain and its associated DNA from either S. solfataricus Orc1-1 or Orc1-3 (PDB code 2qby) were then superposed using the AAA+ domain of Orc4 as a guide. Both dockings indicate that a region of density previously assigned to the Orc6 subunit actually corresponds to the DNA duplex. Although superpositioning of the AAA+ domain of S. solfataricus Orc1-3 onto Orc4 better positions duplex DNA in the observed EM density (than does the comparable exercise using the Orc1-1•DNA complex), the curvature of the DNA (as present in the Orc1-3•DNA co-crystal structure) results in a greater number of clashes between DNA and ORC subunits. Nevertheless, both docking scenarios are consistent with a DNA binding mode of ORC where DNA runs through the central channel. Note that the handedness of the EM map (EMD-5381) has been corrected in this figure because it has been reported that the original handedness was inverted. For clarity, the winged-helix domain of Orc2 is omitted from the remodeled ORC structure in (b). c) The autoinhibited ORC conformation observed in the crystal is, unlike the remodeled Orc1 configuration, similar to the ORC conformation observed in Drosophila ORC EM reconstructions. Docking of the ORC crystal structure (top panels) or the remodeled activated ORC structure (bottom panels) into a prior 3D EM reconstruction of Drosophila ORC (EMD-2479) reveals excellent agreement between EM and crystal structures, but not between EM and modeled activated ORC structures. The poor fit of the remodeled Orc1 conformation into the EM density suggests that the EM structure represents the autoinhibited state of ORC as seen in the crystal, indicating it is the predominant state in solution. See also Supplementary Video 2.
Extended Data Figure 9
Extended Data Figure 9
Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2-7 complex by binding to the ORC winged-helix (WH) domains. A prior model for ORC•MCM2-7 engagement, proposed from an ORC•Cdc6•Cdt1•MCM2-7 cryo-EM structure generated in the presence of DNA (shown in (a), EMD-5625), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in (b), PDB code 1SXJ) to suggest that ORC’s AAA+ domains engage the MCM2-7•Cdt1 complex. However, using the handedness of the EM volume as reported, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis) points toward the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in (a) and (b). The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a gray circle (WA – Walker A, WB – Walker B, RF – arginine finger). c) Docking of the ORC crystal structure (with Orc1 in its remodeled or “activated” conformation) into the cryo-EM map shown in panel (a) reveals that the WH domains of ORC face an MCM2-7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.
Figure 1
Figure 1
Structure of Drosophila ORC. a) Domain organization of ORC subunits. Dashed lines demarcate the ORC core used for crystallization (TFIIB and CTD – transcription factor II-like and C-terminal domains in Orc6; BAH – bromo-adjacent homology domain in Orc1). b) Crystal structure of ORC. Domains of individual subunits are colored as in (a). c) Side view of ORC (surface) highlighting the two-tiered, domain-swapped organization of the ORC body (Orc1 AAA+ and Orc2 WH domains, as well as N-terminal Orc2 residues (built as polyalanine) are not shown). Exploded view (cartoon) showing the packing of WH domains against adjacent subunits’ AAA+ regions.
Figure 2
Figure 2
Eukaryotic ORC and archaeal Orc WH domains. a) WH domain – DNA interactions in archaeal Orc1-1 (PDB code 2qby chain A). The WH domain (grey) uses a helix-turn-helix (HTH, black and white) motif and a β-hairpin wing (cyan) motif to engage DNA (tan surface). b) Disposition of WH domains in ORC. The ORC WH domains (excepting Orc2) form a collar in which the recognition helices (black) are buried and the β-hairpin wings form an exposed portion of the central channel (the Orc1 AAA+ domain is not shown). The HTH motif and β-hairpin wings of ORC subunits are colored as in (a). c) Schematic of the HTH and β-hairpin motifs in ORC.
Figure 3
Figure 3
AAA+/AAA+ domain interactions in ORC. a) Pairwise AAA+ interactions in the Orc2-Orc5 oligomer compared to canonical AAA+ interactions in an ATP-bound DnaA dimer (PDB code 2hcb). Superpositions were carried out using the AAA+ domain of the left-most protomer of a given ORC subunit pair and DnaA homodimer. b-d) Comparison of AAA+ domain assemblies (to scale) for Orc2-5 (b), ATP-assembled DnaA (PDB code 2hcb) (c), and Replication Factor C (RFC, PDB code 1sxj)(d). Upper panels depict side views, with the purple AAA+ domains of DnaA and RFC co-oriented as per the Orc4 AAA+ region. Lower panels show views down the central channel of ORC and RFC, or slightly offset from the helical axis of DnaA. The initiator specific motifs (ISMs, red) of Orc2-5 and DnaA are highlighted (RFC lacks this element). Only the AAA+ domains of ORC, DnaA, and RFC are shown; the RFC-A subunit is omitted. Coloring for ORC is maintained as in panel (a), with sequential subunits in DnaA and RFC colored accordingly.
Figure 4
Figure 4
An unanticipated but naturally-occurring Orc1 conformation. a) The Orc1 AAA+ domain is disengaged from Orc4 and sits above the plane of the Orc2-Orc5 AAA+ oligomer. Only the AAA+ domains of Orc1-5 and the WH domain of Orc1 are depicted; the hinge point between the Orc1 AAA+ and WH regions is indicated. b) The Orc1 conformation is stabilized by contacts (asterisks) between its AAA+ domain and the WH domains of Orc1-3 (the C-terminal helix of Orc1 is in dark blue). See also Extended Data Fig. 6f–h. c) Docking study showing that the ORC crystal structure matches the 3D EM volume of Drosophila ORC (EMD-2479). See also Extended Data Fig. 8c and Supplementary Video 2.
Figure 5
Figure 5
The central channel in ORC likely binds DNA. a) Structural alignment of Orc4 onto DNA-bound S. solfataricus Orc1-1 (PDB code 2qby chain A). b) Reciprocal superposition of DNA-bound archaeal Orc1-1 onto Orc4 in the context of ORC aligns the DNA duplex with ORC’s central channel. The ISMs of Orc2-5 and β-hairpin wings of Orc1 and Orc3-5 (surface) form two semicircular, quasi-spiral collars around the docked DNA (Orc1 AAA+ and Orc2 WH domains are omitted; the remainder of ORC is shown as transparent cartoons). c) The DNA path (tan ribbon) through the central channel is partially blocked by the Orc1 AAA+ (orange cartoon) and the Orc2 WH (green cartoon) domains. The remainder of ORC is shown as surface representation. Coloring per (b).
Figure 6
Figure 6
Model for ORC activation and its functional consequences. a) A ~105° rotation of the Orc1 ATPase fold about a hinge point within the AAA+/WH linker region juxtaposes the Orc1 active site with the arginine finger surface of Orc4 (only Orc1, Orc4 and the Orc5 AAA+ domain are depicted, both as molecular model and schematic). b) Repositioning of Orc1 extends the circuit of ISMs around the prospective DNA binding site of the central channel. The Orc2 WH domain is omitted; DNA is modeled as per Fig. 5 and shown as surface. c) Model for ORC activation and subsequent Cdc6 and MCM2-7 recruitment/loading. i) Prospective recruitment of autoinhibited Drosophila ORC to DNA by elements such as the TFIIB domain of Orc6. ii) Activation of ORC promotes productive Orc1•Orc4 interactions and Orc2 WH domain repositioning, exposing a gap in the ORC ring. iii) DNA binds to ORC’s central channel through ISM/β-hairpin wing interactions, iv) Cdc6 binds between Orc2 and Orc1, trapping DNA inside the complex. v) ORC•Cdc6 binds to and loads one MCM2-7 hexamer (aided by Cdt1) using the WH domain face of ORC•Cdc6. During loading, two MCM2-7 particles are assembled into a double hexamer,
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