Structural Basis for the Activation and Target Site Specificity of CDC7 Kinase

CDC7 is an essential Ser/Thr kinase that acts upon the replicative helicase throughout the S phase of the cell cycle and is activated by DBF4. Here, we present crystal structures of a highly active human CDC7-DBF4 construct. The structures reveal a zinc-finger domain at the end of the kinase insert 2 that pins the CDC7 activation loop to motif M of DBF4 and the C lobe of CDC7. These interactions lead to ordering of the substrate-binding platform and full opening of the kinase active site. In a co-crystal structure with a mimic of MCM2 Ser40 phosphorylation target, the invariant CDC7 residues Arg373 and Arg380 engage phospho-Ser41 at substrate P+1 position, explaining the selectivity of the S-phase kinase for Ser/Thr residues followed by a pre-phosphorylated or an acidic residue. Our results clarify the role of DBF4 in activation of CDC7 and elucidate the structural basis for recognition of its preferred substrates. [Display omitted] •DBF4 activates CDC7 kinase via a two-step mechanism•Zinc-finger domain in CDC7 KI2 interacts with DBF4 motif M•Invariant CDC7 residues Arg373 and Arg380 engage P+1 substrate site CDC7 is a protein kinase that is essential for cell division. Using X-ray crystallography, Cherepanov and colleagues explain the two-step mechanism of CDC7 activation by its dedicated regulator protein DBF4. They also identify amino acid residues in CDC7 that are crucial for the recognition of its preferred substrates.