‘AND’ logic gates at work: Crystal structure of Rad53 bound to Dbf4 and Cdc7

Abstract

Forkhead-associated (FHA) domains are phosphopeptide recognition modules found in many signaling proteins. The Saccharomyces cerevisiae protein kinase Rad53 is a key regulator of the DNA damage checkpoint and uses its two FHA domains to interact with multiple binding partners during the checkpoint response. One of these binding partners is the Dbf4-dependent kinase (DDK), a heterodimer composed of the Cdc7 kinase and its regulatory subunit Dbf4. Binding of Rad53 to DDK, through its N-terminal FHA (FHA1) domain, ultimately inhibits DDK kinase activity, thereby preventing firing of late origins. We have previously found that the FHA1 domain of Rad53 binds simultaneously to Dbf4 and a phosphoepitope, suggesting that this domain functions as an ‘AND’ logic gate. Here, we present the crystal structures of the FHA1 domain of Rad53 bound to Dbf4, in the presence and absence of a Cdc7 phosphorylated peptide. Our results reveal how the FHA1 uses a canonical binding interface to recognize the Cdc7 phosphopeptide and a non-canonical interface to bind Dbf4. Based on these data we propose a mechanism to explain how Rad53 enhances the specificity of FHA1-mediated transient interactions.

The Sir4 H‐ BRCT domain interacts with phospho‐proteins to sequester and repress yeast heterochromatin

“…D Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647), and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and Western blotting with indicated antibodies in the presence and absence of lambda phosphatase. where it is used to bind the FHA1 domain of Rad53 in a phosphorylation-independent manner (Matthews et al, 2012(Matthews et al, , 2014Almawi et al, 2016). Here, we show that Dbf4 H-BRCT also has the capacity to bind to phosphorylated Esc1 and Ubp10 peptides, with nanomolar affinity, as does Sir4 H-BRCT.…”

“…Cells lacking endogenous sir4 and expressing the Myc-tagged H-BRCT domain of Sir4, either wt or carrying mutations (RKR) were subjected to anti-Myc immunoprecipitation followed by LC-MS/MS analyses. where it is used to bind the FHA1 domain of Rad53 in a phosphorylation-independent manner (Matthews et al, 2012(Matthews et al, , 2014Almawi et al, 2016). C MST analysis of the binding interactions between Sir4 H-BRCT and Cy5-labeled phospho (filled circles) and non-phospho-peptides (empty circles) from Tom1 and Cbf1.…”

“…K D represents the equilibrium dissociation constant. Intriguingly, given that the putative phospho-epitope binding region in Dbf4 H-BRCT partially overlaps with the surface patch engaged in FHA1-Rad53 interaction (Almawi et al, 2016), the two binding modes may be mutually exclusive. DF norm [&] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence.…”

“…Further work is needed to determine whether the Dbf4 H-BRCT domain acts as versatile phospho-protein binder, as demonstrated for the Sir4 H-BRCT domain. Intriguingly, given that the putative phospho-epitope binding region in Dbf4 H-BRCT partially overlaps with the surface patch engaged in FHA1-Rad53 interaction (Almawi et al, 2016), the two binding modes may be mutually exclusive. We have characterized biochemically and structurally the Sir4 H-BRCT domain and have shown that it is critical for Sir4's silencing function.…”

“…Nevertheless, we wondered whether Sir4 H-BRCT would also regulate Rad53 activity by binding to the FHA1 domain in a similar way. Recently, the crystal structure of Dbf4 H-BRCT in complex with the FHA1 domain of Rad53 bound to a CDC7 peptide was determined using an engineered single chain chimera of the two proteins revealing a bipartite interaction surface (Almawi et al, 2016). Superposition of Sir4 H-BRCT onto the Dbf4 H-BRCT-Rad53-CDC7 complex ( Fig 2B) identified Dbf4 H-BRCT helix a1 residues contributing to interface I (black dashed circle in Fig 2B) as partially conserved in Sir4, while interface II (blue dashed circle in Fig 2B), mainly formed by the loop connecting Dbf4 H-BRCT helix a3 and strand b4, was not conserved as a seven-residue insert replaces it (orange bar in Fig 2A).…”