Fig. 6. Interaction with the AAA-lid domain of Orc5 by Mcm6 C-WHD is essential for Cdt1 stabilization of half MCM.

a Cryo-EM structure of the OCCM complex⁸. The ORC-Cdc6 ring is illustrated in light grey mesh, while the MCM-Cdt1 ring is in dark grey mesh. The surface of specific individual proteins are highlighted in different colours: Orc 4 (green), Orc5 (blue), Mcm6 (black), and Cdt1 (orange). Of these, the domains studied in this work are further highlighted with their secondary structure, maintaining the colours (extended C-WHD of Cdt1, C-WHD of Mcm6, Orc4 C-WHD and Orc5 AAA lid). Note that the secondary structure of Mcm3 C-WHD is also illustrated in grey. The zoomed area shows in detail the interaction between the Mcm6 C-WHD (in black) and the Orc5 AAA+lid domain (in blue). Side chains of residues studied are depicted in grey (Mcm6) and cyan (Orc5). Specifically, D924 of Mcm6 forms an ionic pair with K286 of Orc5, while M975 of Mcm6 interacts with a hydrophobic patch of Orc5, composed by L242, Q245 and Y285 (Supplementary Fig. 7). In addition, the position of a semiconserved I977 in Mcm6 is also illustrated. The specific amino acid substitutions in Mcm6, containing 2-5 and 2-6 mutations, are detailed in the box. Note that 2-6 mutations include two different mutations already present in 2-1 (Y906 and R972) and in 2-5 mutations (D924 and M975). b PreRC assay with MCM-Cdt1 containing Mcm6 wt, 1-1 and 2-5 mutations. c PreRC assay comparing Mcm6 2-5 and 2-6 mutations. Additionally, Mcm6 2-1 is also included because some mutations present in Mcm6 2-6 are also present in Mcm6 2-1. d Alignment of the residues involved in the interaction between the Mcm6 C-WHD and the Orc5 AAA+-lid. The ionic pairs between Mcm6 D924 and Orc5 K286 are highlighted in red asterisks. The structural conservation between the hydrophobic patch of Orc5 (L242, Q245 and Y285) and Mcm6 M975 is highlighted in blue asterisks. Sc Saccharomyces cerevisiae, Dm Drosophila melanogaster, Xl Xenopus laevis, Dr Danio rerio, Mm Mus musculus, Hs Homo sapiens.