Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

Abstract

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.

Intermolecular Binding between TIFA-FHA and TIFA-pT Mediates Tumor Necrosis Factor Alpha Stimulation and NF-κB Activation

“…The model for the aggregation of TIFA therefore uncovers a new mechanism for FHA domain functions: the unphosphorylated FHA domain-containing protein exists as an intrinsic dimer that oligomerizes via the intermolecular FHA-pT bindings between dimers. Interestingly, the FHA domains of both human and mouse MDC1 (mediator of DNA damage checkpoint 1) proteins were recently shown to exist as intrinsic dimers in solution and in crystals (13,17,28).…”

Dual recognition of phosphoserine and phosphotyrosine in histone variant H2A.X by DNA damage response protein MCPH1

“…It thus appears that tipping the concentration balance in favor of MCPH1 or MDC1 could significantly alter the composition at γH2A.X nucleosomes. Also, the ability of MDC1 to dimerize in response to DNA damage may considerably change its capacity to bind γH2A.X with avidity effects coming into play (36,37). This may explain how MDC1 independently engages γH2A.X in the presence of MCPH1.…”

Dimerization Mediated by a Divergent Forkhead-associated Domain Is Essential for the DNA Damage and Spindle Functions of Fission Yeast Mdb1

“…4, A and B). As a comparison, the K d value is 9.2 M for unphosphorylated MDC1 FHA dimer, 32 nM for MDC1-FHA dimer with a phosphorylated Thr-4, and 0.19 nM for MU2-FHA dimer (10,12). The different stabilities of these FHA dimers are correlated with the sizes of their dimer interfaces.…”

“…The samples were centrifuged at 25°C with 25,000, 30,000, and 38,000 rpm and detected using absorbance at 280 nm. All data were globally fit using a self-association model in SedPhat program as described previously (10).…”

“…The structures revealed that MDC1-FHA has an intrinsic ability to form a homodimer. Furthermore, an intersubunit interaction between MDC1-FHA and a phosphorylation site near the extreme N terminus of MDC1 enhances the dimerization (10,11). The exact physiological function of the dimerization of MDC1 remains unresolved, because two studies showed that loss of dimerization partially impairs the ability of MDC1 to localize to DNA damage sites (8,10), whereas another study concluded that dimerization does not contribute to MDC1 accumulation at sites of DNA breaks (11).…”