Zhou H et al. (2021), Structure of CRL2Lrr1, the E3 ubiquitin ligase ...

XB-ART-58677

Nucleic Acids Res 2021 Dec 16;4922:13194-13206. doi: 10.1093/nar/gkab1174.

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Structure of CRL2Lrr1, the E3 ubiquitin ligase that promotes DNA replication termination in vertebrates.

Zhou H , Zaher MS , Walter JC , Brown A .

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When vertebrate replisomes from neighboring origins converge, the Mcm7 subunit of the replicative helicase, CMG, is ubiquitylated by the E3 ubiquitin ligase, CRL2Lrr1. Polyubiquitylated CMG is then disassembled by the p97 ATPase, leading to replication termination. To avoid premature replisome disassembly, CRL2Lrr1 is only recruited to CMGs after they converge, but the underlying mechanism is unclear. Here, we use cryogenic electron microscopy to determine structures of recombinant Xenopus laevis CRL2Lrr1 with and without neddylation. The structures reveal that CRL2Lrr1 adopts an unusually open architecture, in which the putative substrate-recognition subunit, Lrr1, is located far from the catalytic module that catalyzes ubiquitin transfer. We further demonstrate that a predicted, flexible pleckstrin homology domain at the N-terminus of Lrr1 is essential to target CRL2Lrr1 to terminated CMGs. We propose a hypothetical model that explains how CRL2Lrr1's catalytic module is positioned next to the ubiquitylation site on Mcm7, and why CRL2Lrr1 binds CMG only after replisomes converge.

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Species referenced: Xenopus laevis
Genes referenced: cul2 eif4g2 lrr1 mcm3 mcm5 mcm7 rbx1 timeless tipin usp2 vhl
GO keywords: DNA replication [+]

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	Figure 1. Structure of CRL2Lrr1. (A) Two views of the cryo-EM map of CRL2Lrr1 (State 1) with each of the five subunits uniquely colored. The site of Cul2 neddylation is marked with an asterisk. (B) Atomic model of CRL2Lrr1 derived from the cryo-EM map.
	Figure 2. Interaction between Lrr1 and Cul2:EloBC. (A) Domain architecture of X. laevis Lrr1, showing the sequence of the BC and Cul2 boxes. (B) Atomic model of Lrr1. The model of the PH domain is predicted by AlphaFold, whereas the models of the LRR, VHL box and zinc finger domains were derived from the experimentally determined cryo-EM map. The dashed line indicates a flexible linker. (C) Overview of the interactions that Lrr1 makes with Cul2 and EloC. (D) Details of the interaction between the Lrr1 BC box and EloC. (E) Details of the interaction between the Lrr1 Cul2 box and Cul2 and EloC. (F) Sequence alignment of the Cul2 α5–α6 loop that interacts with the LRR domain of Lrr1. (G) Organization of the Lrr1 zinc finger showing tetrahedral coordination of a zinc cation. (H) The cysteine residues of the zinc finger (highlighted in yellow) are conserved in vertebrates and flies but absent in Caenorhabditis elegans.
	Figure 3. CRL2Lrr1 adopts an open conformation. (A) Surface representation of an atomic model of the unneddylated CRL2pVHL complex generated by docking two crystal structures (PDB 4WQO and PDB 5N4W) (33,34). (B) Surface representation of the atomic model of the neddylated CRL1β-TRCP–UBE2D–Ub–substrate complex (PDB 6TTU) (30) in a closed conformation, in which the substrate-recognition subunit β-TRCP makes direct contacts with the E2–Ub conjugate. (C) Cryo-EM map of neddylated CRL2Lrr1 colored by subunit. The structure forms an open conformation with the LRR domain of Lrr1 ∼65 Å from the RING domain of Rbx1 that catalyzes ubiquitin transfer even at their closest point. The WHB domain of Cul2 is not visible in the cryo-EM map.
	Figure 4. The PH domain of Lrr1 is crucial for CRL2Lrr1 function. (A) The Lrr1 PH domain is essential for Mcm7 polyubiquitylation, CMG unloading and CRL2Lrr1 recruitment to the replisome in egg extracts. Plasmid DNA was replicated in the indicated extracts in the presence or absence of p97i. After 45 min, the plasmid was recovered, and samples were processed for immunoblotting with the indicated antibodies. Histone H3 was used as a loading control. (B) The Lrr1 PH domain is required for CMG polyubiquitylation in the absence of DNA. rCMG was added to the indicated egg extracts containing p97i and His-tagged ubiquitin (His-Ub). After 40 min, His-Ub was recovered, and samples were immunoblotted with Mcm7 antibody. (C) The Lrr1 PH domain is required for CMG polyubiquitylation in a reconstituted system. rCMG or rCMGK27/28R was preincubated with neddylated or unneddylated rCRL2Lrr1 or rCRL2Lrr1ΔPH, as indicated. Reactions were initiated by the addition of a ubiquitin master mix containing E1, E2 and ubiquitin, followed by incubation at 37°C. Usp2, a deubiquitinating enzyme, was added to confirm that the shifted Mcm7 species were indeed ubiquitylated. Samples were immunoblotted with the indicated antibodies.
	Figure 5. Proposed model for the recruitment of CRL2Lrr1 to postsynthesis replisomes. (A) A schematic of a human replisome subcomplex containing CMG and And-1 (PDB 6XTX) (29), the Tipin and Timeless subunits of the fork protection complex predicted by AlphaFold 2 (25) and replication fork DNA (PDB 6SKL) (16). The leading strand template of the replication fork DNA passes through the channel of CMG, whereas the lagging strand template (indicated with a dashed line) is excluded from the channel between zinc finger domains of Mcm3 and Mcm5. The distance between the ubiquitylation sites (shown in red) and the interface of Mcm3 and Mcm5 ZFs is ∼65 Å. (B) Proposed model showing how CRL2Lrr1 might engage replisomes that have translocated onto dsDNA. The catalytic module is positioned near the ubiquitylation site for efficient ubiquitin transfer and the LRR domain of Lrr1 engages the zinc finger domains of Mcm3 and Mcm5. The PH domain of Lrr1 could potentially bind And-1, CMG, the fork protection complex or DNA.

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