Abstract
The molecular mechanisms by which the human CDC25B activates the CDK1/cyclin B complex in the cell cycle, as well as how it can be inhibited by synthetic inhibitors at the atomic level, are still under investigation. Valuable insights have been gained from the molecular structure here-described, which captures for the first time the interaction between the C-terminal domain of the inactive mutant CDC25B C473S (CDC25B-S) and the commonly used synthetic substrate 3-O-methylfluorescein phosphate (3-OMFP). Crystallographic studies reveal that 3-OMFP engages multiple residues within the active site and the adjacent "swimming pool" of CDC25B-S, establishing specific interactions and prompting local adjustments in this region. These structural features explain the increased resistance to thermal denaturation of CDC25B-S observed through circular dichroism measurements upon substrate binding. The structural changes induced by 3-OMFP lead to a conformation comparable to that of CDC25A bound to its substrate, the CDK2/cyclin A complex. These findings qualify 3-OMFP as a promising starting model for the rational design of selective competitive inhibitors of CDC25B having reduced off-target effects.