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. 2019 Nov 14;48(1):421–431. doi: 10.1093/nar/gkz1037

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Key residues forming Motif I and II of CtCfp1 RID are important for RbBP5 binding. (A) RbBP5/WDR5 complex interacts with Cfp1 RID ZnF. GST-tagged Cfp1 proteins (wild-type or mutants) were incubated with the RbBP5/WDR5 complex. Bound proteins were resolved on a 15% SDS-PAGE and Coomassie-stained. Star (*) indicates a degradation product of GST-tagged Cfp1. The residues located on motif I and Motif II are colored in pink and beige, respectively. (B) Isothermal titration calorimetry curve of Cfp1 RID titrated into RbBP5 β-propeller. (C) ITC values for Cfp1 RID wild-type and mutants. Titrations were performed in duplicate. S.D. represents the standard deviation between the two experiments. N.A.: Experiments could not be performed as the mutant aggregated during ITC. N.B.: No binding could be detected. (C) Replacement of metal coordinating residues lowers the interaction between Cfp1 and RbBP5. Immunoprecipitation of ectopically expressed FLAG-tagged constructs of Cfp1 wild-type and motifs I and II mutants. Their corresponding residues in Chaetomium thermophilum (Ct) are labeled. RbBP5 and FLAG-tagged Cfp1 were detected with indicated antibodies. (D) Cfp1 RID ZnF is evolutionary conserved. A protein sequence alignment of Cfp1 RID domain from Chaetomium thermophilum (Ct) with the corresponding RID domains of Saccharomyces cerevisiae (Cps40), Drosophila melanogaster (Dm), Danio rerio (Dr), Homo sapiens (Hs). RID α-helices are depicted as cylinders and the motifs I/II are highlighted as lines with colors corresponding to the Figure 3. Evolutionary conserved residues are rendered with Blosum62 color scheme. Residues matching the consensus sequence residue at that position are colored in dark blue. Based on the conservation score calculated by Bloosum62 matrix, other residues are coloured in different shades of blue. The black and blue * indicates the residues mutated on CtCfp1 (A) and HsCfp1 (C) respectively.