Crystal Structure of Arabidopsis thaliana Dawdle Forkhead-Associated Domain Reveals a Conserved Phospho-Threonine Recognition Cleft for Dicer-Like 1 Binding

The crystal structure of the Arabidopsis Dawdle (DDL) forkhead-associated (FHA) domain resembles a canonical FHA domain architecture that recognizes a phosphorylated residue. The arrangement of contacting residues in the crystal packing, together with the functional studies, reveals the mechanism of interaction with DCL1.Dawdle (DDL) is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated (FHA) domain at the carboxyl-terminus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7- resolution. DDL FHA structure displays a seven-stranded -sandwich architecture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthamiana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr3(Ile/Val/Leu/Asp) motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated proteinprotein interaction in plant miRNA processing.