Fig. 1: Cyclin B1^mut is capable of activating CDK1, but deficient in separase binding.

A Electrostatic surface potentials of human cyclin B1^wt (top, PDB: 7NJ0) and cyclin B1^mut (bottom, modeled) are illustrated. Close-up views of the cyclin B1 phosphate-binding pocket in cyclin B1^wt and in cyclin B1^mut bound to phosphoserine 1126 of human separase. Mutations of three critical residues (R307E, H320F, K324E) in the binding pocket lead to a complete charge reversal. Electrostatic potentials are contoured from -10 (red) to +10 kTe^-1 (blue). B Sequence conservation of cyclin B1 mapped onto the human cyclin B1 structure (PDB: 7NJ0²⁹). The cyclin B1 structure is shown as ribbon (left) or surface representation (right). Residues that form the phosphate-binding pocket are displayed as sticks in the ribbon representation, and the phosphate-binding pocket is indicated with a dashed circle on the surface representation. The AL2CO program⁸⁹ has been used to map the conservation indices from a multiple sequence alignment of 2000 different cyclin B homologs (including yeast and human) onto the spatial structure of human cyclin B1. Variable regions are shown in cyan, conserved regions in maroon. Bottom, sequence alignment of the pocket region for different cyclins. Sequence comparison of human B-type cyclins with cyclin A2 (top four rows). Sequence conservation of the binding pocket in B1-cyclins across different species (middle six rows). Last row shows the sequence of the designed cyclin B1^mut pocket. Residue numbers for each species are labeled on the right. H.s. (Homo sapiens), M.m. (Mus musculus), R.n. (Rattus norvegicus), B.t. (Bos taurus), D.m. (Drosophila melanogaster), X.l. (Xenopus laevis), S.c. (Saccharomyces cerevisiae). C Radioactive in vitro kinase assay using MBP-CDK1/cyclin B1^wt/mut-His and as substrate MBP-Emi2^NT (aa 1-350) containing a single CDK1 consensus site. At indicated timepoints after reaction start, samples were immunoblotted for MBP and cyclin B1 (α-cyc B1) and analyzed by autoradiography (³³P). CBB (Coomassie brilliant blue). D Quantification of n = 3 independent in vitro kinase assays. Shown are mean values with standard deviations. E Immunoblot analyses of anti-mNG immunoprecipitation (IP) samples of HeLa cells expressing mNG-cyclin B1^wt/mut. Doxycycline treated cells were enriched in mitosis by treatment with 333 nM nocodazole for 20 h. Shown are representative input (IN), flow through (FT) and IP samples. p150 served as IP control.