Abstract
This work focuses on the structural basis of the FHA domain (forkhead-associated domain) in the interaction between MDC 1 (mediator of DNA damage checkpoint 1) and CHK2 (checkpoint kinase 2) upon DNA damage. The MDC1 protein functions as a key mediator that interacts with multiple proteins involved in DNA damage response (DDR) pathway. It binds to not only sensor proteins like ATM and MRE11, but also effector proteins such as CHK2. The complicated phospho-signaling network controls the sensing, initiating, and mediating steps that lead to downstream repair pathways, cell-cycle checkpoints, and apoptosis. Previously, the CHK2 binding site for MDC1-FHA was shown to be pThr68 (the same site recognized by the FHA domain of CHK2 for dimerization and activation of CHK2). To elucidate the molecular mechanism of MDC1-CHK2 interaction, we solved crystal structures of mouse MDC1-FHA and its complex with a human CHK2 peptide containing pThr68. Surprisingly, MDC1-FHA exists as an intrinsic dimer in solution and in crystals. Structural and binding analyses support the pThr+3 ligand specificity of FHA domains, and provide structural insight into MDC1-CHK2 interaction. In order to test whether the dimerization of MDC1-FHA directs MDC1 function in vivo, we selected different MDC1-FHA mutants with disrupted dimerization while maintaining the pThr-binding ability. The full-length MDC1 protein containing such mutations not only failed to dimerize in vivo as suggested by split-GFP system, but also failed to rescue cellular radio-sensitivity caused by MDC1 knockdown. In addition, our result shows that the dimeric feature affects the MDC1 protein turnover rate on DNA lesion sites by which the accurate DNA damage signal can be executed. It implies that the dimeric feature may play a role of super-scaffold to interact with other proteins after DNA damage. In addition, dimerization-dependent trans autophophorylation is a common mechanism to active kinase. Activated ATM kinase phosphorylates CHK2 on Thr68 to trigger CHK2 activation in DNA damage. The pThr68 interacts with its FHA domain, leading to dimerization and T-loop autophosphorylation. However, the molecular basis of this activation process remains unclear. Here we use site-specifically pThr68 CHK2 for biophysical characterization by AUC and provide structural explanation by SAXS analyses. The results show that pThr68 plays a critical role to stabilize CHK2 dimerization. The SAXS results show that pThr68-mediated dimerization is possible to bring two kinases to correct orientation for efficient activation loop trans autophosphorylation.