Abstract
Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. The FHA domain is often part of the multi-domain domain proteins that facilitates the assembling of signaling complex and connecting of the signaling pathway. It has diverse functions in a protein and plays major roles in jodyad53 and TIFA. (i) Rad53: The budding yeast homolog of Checkpoint kinase 2. Rad53 controls the S-phase checkpoint as well as the G1 and G2 DNA damage checkpoints. The kinase activity of Rad53 is dependent on its autophosphorylation, which is supported by dimerization. The open and closed intermediate conformations of Rad53 kinase domain were revealed through X-ray crystallography using Rad532-466 (consist of SCD1, FHA1 and Kinase domain: Rad53SFK). The open and closed forms of Rad53SFK differ in the relative rotation and movement of αC helix in the N-lobe. In the open form, the αC helix is further away from the β3 strand. In the closed form, the αC helix is closer to the β3 strand and the E244 on the αC helix forms the conserved salt bridge with K227 on the β3 strand, which has been shown to be crucial for the stable binding with ATP. In addition, the SAXS analyses of Rad53SFK and phospho-Rad53SFK showed that the Rad53SFK has various conformations in solution while the FHA domains and kinase domain of phospho-Rad53SFK are in close proximity in solution. The results provided important insights into how the autophosphorylation mechanism of Rad53 is regulated by phospho-priming induced dimerization, and why proximity driven by dimerization is distinct from that induced by concentration. (ii) TIFA: TRAF-interacting protein with a FHA domain. TIFA is involved in the activation of NF-κB. Upon stimulation, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization. The oligomeric form of TIFA could promote TRAF6 oligomerization and ubiquitination, which activates NF-κB. The structural information of TIFA (tTIFA1-150) and its complex with TIFA N-terminal phosphorylated Thr9 peptide 1-15 (pThr9-TIFA1-15) showed the unique structure and the novel binding mode of TIFA FHA domain. The intrinsic TIFA dimer has a distinct dimer interface, and its structural features revealed that pThr9-FHA domain binding is between inter-dimers rather than intra-dimers. The structural information helped us gained insight into the TIFA dimer, and uncovered the molecular mechanism for the functionally important oligomerization of TIFA.