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

ABSTRACT: 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.

SUBMITTER: Dick SD

PROVIDER: S-EPMC7416108 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Dick Samual D SD Federico Stefania S Hughes Siobhan M SM Pye Valerie E VE O'Reilly Nicola N Cherepanov Peter P

Structure (London, England : 1993) 20200609 8

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 c ...[more]

PMID: 32521228

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Project description:Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg(2+)-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, alpha and beta, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKbeta encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GKalpha. The crystal structures of recombinant GKalphabeta and GKbetabeta from Namalycastis sp. were determined at 2.6 and 2.4 A resolution, respectively. In addition, the structure of the GKbetabeta was determined at 2.3 A resolution in complex with a transition state analogue, Mg(2+)-ADP-NO(3)(-)-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GKalphabeta are structurally defined, and the longer N-terminus of the beta subunit is anchored at the dimer interface. In GKbetabeta the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GKalphabeta amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely alpha or solely beta chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

Dataset Information

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

Publications

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

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