Mechanistic Insights into Phosphoprotein-Binding FHA Domains

Abstract

CONSPECTUS FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is: how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a β-sandwich of two β-sheets. The conserved Arg and Ser of the recognition loops recognize the phosphorylation of the Thr targeted. Side chains emanating from loops that join β-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphoThr in sequence (pT+3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT+3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged, polar side chains at pT+3. However, another FHA domain instead chooses on the basis of pT−2, pT−3, and pT+1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a β-strand to its β-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphoThr peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the β-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

Molecular Basis of BACH1/FANCJ Recognition by TopBP1 in DNA Replication Checkpoint Control

“…Interestingly, this recognition of the Thr(P) side chain is similar to that employed by FHA domains, which specifically bind Thr(P) phospho-peptides through conserved arginine residues (65,66). For example, Arg 61 of the RNF8 FHA domain makes a bidentate interaction with the phosphate of its target Thr(P), whereas Arg 42 mediates an interaction between the phosphate group and Ϫ2 of the peptide backbone.…”

Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases

“…The importance of the +3 position in the common motif, which is typically a critical position for FHA recognition (24), suggests that phospho-dependent interactions of STPK substrates with FHA domain proteins may also contribute to specific downstream signaling mediated by these kinases. In support of this idea, the GarA FHA domain was previously shown to prefer hydrophobic residues at the +3 position relative to phospho-Thr (25).…”

The Intrinsically Disordered N-terminal Region of AtREM1.3 Remorin Protein Mediates Protein-Protein Interactions

“…These data are in agreement with previous reports (54 -57), which describe that phosphorylation clusters preferentially within ID regions and often mediates protein interactions. For example, 14-3-3 proteins can discriminate between phosphorylated and non-phosphorylated partners (58). Interaction of a 14-3-3 protein with the RNA-binding K-homology splicing regulator protein (KSRP) is promoted by phosphorylation of the latter, which drives relocalization of KSRP into the nucleus, where it performs its mRNA degrading activity (59).…”