Interactions of forkhead-associated domain, FHA1 of Saccharomyces cerevisiae Rad53 kinase with itself and the biological partners, Mdt1 AND Rad9

The forkhead-associated (FHA) domain is a universal protein-protein interaction motif which plays a critical role in regulating cancer and cell death by interacting with other proteins. The aim of this research is to determine its real binding specificities and the molecular basis of its binding to a diverse set of ligands. This in-turn, could explain its diverse biological functions in cell. The FHA domain used in this research is from the Rad53 kinase protein (FHA1) in Saccharomyces cerevisiae which is functionally similar to the human Chk2-kinase, a pivotal cell cycle protein. The FHA domains of both kinases, when triggered by DNA damage, interact with downstream phosphorylated proteins to coordinate checkpoint signaling pathways.

In this study, a solution structure of the FHA1 domain in complex with a biologically relevant binding peptide from protein Mdt1, which functions downstream of Rad53 kinase, has been determined. Nuclear Magnetic Resonance (NMR) spectroscopy, using Isotope-filtered Nuclear Overhauser Effect (NOE) experiments, was used to determine the points of contacts across the binding interface of this protein-peptide complex. This structure is significantly different from numerous structures solved with synthetic peptides and casts doubt on previously accepted interactions deduced from other well established chemical methods. Our study on the elongated versions of Mdt1 peptide and its interactions with FHA1 domain indicates the lack of extensive binding for recognition and specificity.

At a functional level, the Rad53 kinase gets activated by DNA damage leading to its oligomerization and autophosphorylation. This study indicates that FHA1 recognizes a phosphorylated peptide from its own N-terminal SCD (SQ/TQ Cluster Domain) with a "different" binding motif (pTQ). NMR experiments were performed to compare the affinity of FHA1 and FHA2 domains with this peptide. FHA1 domain, instead of FHA2 domain, has been shown to be responsible for Rad53 kinase's dimerization and consequent phosphorylation. Furthermore, the possibility of obtaining a phosphorylated FHA1 domain to study the protein-protein interactions involving other proteins like Dun1 has been explored. In conclusion, this study shows that FHA domain recognition is dictated to some extent by its function and not purely by the sequence of binding peptides as predicted by earlier reports.