Phosphoprotein-binding Domains Research Articles

The FHA domain mediates phosphoprotein interactions

The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first identified in a group of forkhead transcription factors but is present in a wide variety of proteins from both prokaryotes and eukaryotes. In yeast and human, many proteins containing an FHA domain are found in the nucleus and involved in DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain is part of a protein that is localized to the plasma membrane and participates in the regulation of receptor-like protein kinase signaling pathways. Recent studies show that a functional FHA domain consists of 120-140 amino acid residues, which is significantly larger than the sequence motif first described. Although FHA domains do not exhibit extensive sequence similarity, they share similar secondary and tertiary structures, featuring a sandwich of two anti-parallel (beta)-sheets. One intriguing finding is that FHA domains may bind phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing them from other well-studied phosphoprotein-binding domains. The diversity of proteins containing FHA domains and potential differences in binding specificities suggest the FHA domain is involved in coordinating diverse cellular processes.

II. Structure and specificity of the interaction between the FHA2 domain of rad53 and phosphotyrosyl peptides †,1

The forkhead-associated (FHA) domain is a protein module found in many proteins involved in cell signaling in response to DNA damage. It has been suggested to bind to pThr sites of its target protein. Recently we have determined the first structure of an FHA domain, FHA2 from the yeast protein Rad53, and demonstrated that FHA2 binds to a pTyr-containing peptide 826EDI(pY)YLD 832 from Rad9, with a moderate affinity ( K d ca. 100 μM). We now report the solution structure of the complex of FHA2 bound with this pTyr peptide. The structure shows that the phosphate group of pTyr interacts directly with three arginine residues (605, 617, and 620), and that the leucine residue at the +2 position from the pTyr interacts with a hydrophobic surface on FHA2. The sequence specificity of FHA2 was determined by screening a combinatorial pTyr library. The results clearly show that FHA2 recognizes specific sequences C-terminal to pTyr with the following consensus: XX(pY)N 1N 2N 3, where N 1 = Leu, Met, Phe, or Ile, N 2 = Tyr, Phe, Leu, or Met, and N 3 = Phe, Leu, or Met. Two of the selected peptides, GF(pY)LYFIR and DV(pY)FYMIR, bind FHA2 with K d values of 1.1 and 5.0 μM, respectively. The results, along with other recent reports, demonstrate that the FHA domain is a new class of phosphoprotein-binding domain, capable of binding both pTyr and pThr sequences.