|Rad53, a key checkpoint protein in Saccharomyces cerevisiae, has a central catalytic kinase domain as well as two forkhead-associated (FHA) domains, FHA1 and FHA2. Along with the extensively studied phosphothreonine binding pocket, characteristic of all FHA domains, the Duncker lab in collaboration with Alba Guarné’s lab, recently reported that FHA1, but not FHA2, has an additional conserved region on one of its lateral surfaces. Mutation of this novel FHA1 motif abrogated the interaction between Rad53 and Dbf4 (the regulatory subunit of the Dbf4-Cdc7 kinase complex), and rendered cells sensitive to genotoxic stress. When corresponding residues are mutated in Dun1 and Mek1 (other S. cerevisiae FHA domains), affinities for the Rad53 FHA1 ligand Dbf4 can be altered, highlighting the importance of the region. Additionally, structural and biochemical data has shown that several previously unidentified residues on Dbf4 are important for the interaction with the lateral surface of FHA1.
I have since investigated whether this non-canonical FHA1 interaction surface mediates binding to other Rad53 ligands, and have found that it has a variable requirement. FHA1 has numerous binding partners in the cell, each with distinct cell cycle progression and checkpoint roles. While the same FHA1 lateral domain mutants with a disrupted Dbf4 interaction retained wild type affinity for Sgs1 and Cdc7, an intermediate effect was observed with Sld3. Taking this data as a whole, it shows that this lateral region of FHA1 is specific for mediating the interaction with Dbf4, and mutations of this region do not disrupt binding to other FHA1 ligands.
Through bioinformatics analysis, several novel conserved regions in numerous other proteins with FHA domains in S. cerevisiae, including Dun1 and Mek1, have been identified. Similar to Rad53 FHA1, mutation of conserved residues in the novel Dun FHA motif disrupted its interaction with Sml1, a Dun1 ligand. Furthermore, these mutants showed wild type levels of interaction when interacting with the known Dun1 ligand Rad53. Spotting plate assays were conducted with two genotoxic agents: hydroxyurea (HU) and methyl methanesulfate (MMS). A moderate increased sensitivity was seen on the HU plates for the Dun1 FHA lateral surface mutants, however cells were not completely sensitive. This work highlights the importance of newly identified FHA domain motifs in mediating non-canonical protein-protein interactions.