NMR and mutation studies of the interaction between Mcm6-CBD and Cdt1-MBD. A, a part of the overlaid ¹H-¹⁵N-HSQC spectra of ¹⁵N-labeled CBD in free form (black) with that titrated with non-labeled GB1-Cdt1 410–445 (red) at a molar ratio of 1:4. Residues that undergo significant changes in chemical shifts upon formation of the complex with Cdt1-MBD are highlighted by arrows and labeled with peak assignments. B, chemical shift differences between the free-form and MBD saturated CBDs. C, chemical shift perturbations in the presence of MBD are colored onto the structure of CBD in ribbon representation. Residues with chemical shift perturbations ranging from 0.04 to 0.12 ppm are colored in green, whereas residues with chemical shift perturbations larger than 0.12 ppm are shown in red. Key residues, Glu-757, Glu-763, and Leu-766, are shown in the stick model. D, surface presentation of CBD with residues showing chemical shift perturbations as described in B. E, the effect of a single-point mutation in Mcm6-CBD on in vitro Cdt1-MBD interaction. The upper panel indicates a relatively equal amount of GST-Mcm6-CBD mutant proteins used in the GST pulldown assay. The immunoblot with anti-His antibodies shows the binding of GB1-His-MBD to the wild-type (wt) or mutant CBD (lower panel). Mutants of CBD highlighted in red exhibited a significant reduction in binding to Cdt1-MBD.

In vitro interaction of Mcm6-CBD with Cdt1-MBD and solution structure of CBD of human Mcm6. A, the C terminus of Mcm6 interacts with the C-terminal region of Cdt1. Thioredoxin (Trx)-Cdt1-(392–471) was pulled down by GST-Mcm6-(708–821) and visualized by Coomassie Blue staining. The asterisk indicates the Cdt1 degradation product. B, the minimal region of Cdt1 required for Mcm6 interaction. GB1-Cdt1-(410–445) was pulled down by GST-Mcm6-(748–821) or GST-Mcm6-(708–821) and visualized by Coomassie Blue staining. C, the left panel shows the backbone superposition of the 20 lowest energy NMR structures of Mcm6-CBD. Secondary structural elements are indicated by color coding: α-helices (red), β-strands (green), and loops (cyan, gray). N-terminal and C-terminal ends are indicated as N and C. The right panel shows a ribbon representation of the same structure of CBD using the coordinates of the lowest energy structure. D, the {¹H}-¹⁵N heteronuclear NOEs of each amino acid are depicted. Secondary structural elements are indicated with color coding as in B on the top of the figure. E, the structure of the hydrophobic cage of CBD is shown with the side chains of important residues such as Leu-726 and Leu-748 presented in green and residues Ile-723, Ile-772, and Ile-773 shown in red.