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

Abstract

Summary 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.

The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer

“…3d ). The zinc-binding site formed by yeast and human Dbf4 proteins is highly conserved and helps to keep the Cdc7 αC helix and kinase insert 2 in their “kinase active” state, as described in the context of human Cdc7 41 (Fig. 3e, f ).…”

“…3e, f ). Even though residues forming a second zinc-binding site are not conserved between human and yeast Cdc7, a zinc ion is observed in a nearby alternative location relative to the structure of the human Cdc7, and may therefore stabilise a similar section of the kinase 41 .…”

“…The activation loop region is defined by the start of the DFG motif and the end of APE motif, with the kinase insert 2 located in-between the two motifs 41 . Here, we have resolved large sections of the activation loop, which makes direct contacts with Dbf4 and Mcm4 and may contribute to anchoring the activation loop in the active site (Fig.…”

“…Inhibition of DDK activity causes apoptosis in cancer cells, but not in normal cells, and therefore Cdc7 is seen as an attractive therapeutic target. Indeed, crystal structures of the human Cdc7 core in complex with small conserved regions of Dbf4 40 , 41 have led to a number of drug discovery projects and currently several inhibitors are in various stages of clinical development 6 , 42 , 43 .…”

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

“…Dbf4 contains three evolutionarily conserved sequence motifs, termed N, M, and C that constitute parts of distinct domains separated by flexible linkers ( Hughes et al, 2012 ; Masai and Arai, 2000 ). The M and C domains embrace the Cdc7 kinase to mediate Cdc7 activation, while the N motif forms part of an N-terminal BRCT domain that is dispensable for Cdc7 activation ( Almawi et al, 2016 ; Dick et al, 2020 ; Hughes et al, 2012 ). Intriguingly, an N-terminal fragment encompassing the BRCT and M domains is required and sufficient to target Dbf4 to MCM complexes loaded at replication origins ( Dowell et al, 1994 ; Francis et al, 2009 ), while a physical interaction between Rad53 and the Dbf4 BRCT domain has been proposed to contribute to checkpoint-dependent origin control ( Chen et al, 2013 ; Duncker et al, 2002 ; Matthews et al, 2012 ; Matthews et al, 2014 ).…”