FHA Domains

Figure 1 14-3-3/phosphopeptide interactions. Dimerization of two 14-3-3 monomers, each of which is composed of nine a-helices, forms a cleft within which phosphoserine-containing ligands bind (shown in ball-and-stick representation). A single, multiply phosphorylated protein ligand may bind simultaneously to both available sites. Alternatively, two singly phosphorylated proteins can bind, one to each monomer, allowing 14-3-3 to act as a molecular scaffold for the assembly of diverse signaling complexes [9,24]. Reprinted from Yaffe and Smerdon [75], with permission.

catalytic activity, as observed, for example, with Raf, exoenzyme S, and serotonin N-acetyltransferase [12-14]. For others, 14-3-3 appears to regulate interactions between its bound protein and other molecules within the cell. For example, growth-factor-mediated phosphorylation of the pro-apoptotic Bcl-2 family protein BAD at Ser-136 and/or Ser-112 facilitates its interaction with 14-3-3 proteins and blocks its association with anti-apoptotic Bcl-2 family members at the mitochondrial membrane [15-17]. Finally, in a number of cases, 14-3-3 proteins appear to play an important role in controlling the subcellular localization of bound ligands such as Cdc25 and Forkhead family transcription factors [18-21].

The X-ray structures of 14-3-31 and Z revealed that the molecule is a cup-shaped dimer [22,23] in which each monomeric subunit consists of nine a-helices (Fig. 1). The dimer interface is formed from helices aA, aC, and aD, creating a 35-A x 35-A x 20-A central channel where binding to peptide and protein ligands occurs. Ligand-bound 14-3-3 structures have shown that peptides bind within an amphipathic groove along each edge of the central channel [9,24,25] with the entire phosphopeptide main chain in a highly extended conformation until two residues after the pSer, when there is a sudden sharp change in peptide chain direction required to exit the 14-3-3 binding cleft. More recently, the structure of 14-3-3Z bound to a bona fide protein ligand, serotonin N-acetyltransferase, has been solved [26]. In this structure, the 14-3-3 binding portion of the enzyme displays a conformation very similar to that seen in isolated phosphopeptide:14-3-3 complexes, including the extended conformation and sudden alteration in chain direction. Furthermore, 14-3-3-binding at least partially restructures the substrate binding site on serotonin N-acetyltransferase, rationalizing some of the 14-3-3 effects on enzyme activity. Additional X-ray structures of 14-3-3-bound complexes are required before a detailed mechanistic understanding of 14-3-3 function emerges.

Forkhead-associated (FHA) domains are a recently recognized pThr-binding module found in several prokaryotic and eukaryotic proteins including kinases, phosphatases, transcription factors, kinesin-like motors, and regulators of small G proteins. FHA domains are «140 amino acids in length and extend significantly beyond the core homology region first identified by sequence profiling [27-33].

Recognition that FHA domains were pThr-binding modules came from findings that the FHA domain of KAPP, a protein phosphatase in Arabidopsis, was critical for its interaction with phosphorylated receptor-like kinases [28,34] and that the FHA domains within the Saccharomyces cere-visiae cell-cycle checkpoint kinase Rad53p were essential for interaction with the phosphorylated DNA damage control protein Rad9p [35,36]. Durocher et al. [36] were the first to demonstrate that FHA domains could bind directly to short pThr-containing peptides in isolation. Data regarding the specificity of different FHA domains for pThr-based sequence motifs comes from peptide library experiments that show sequence-specific binding involving amino acids from the pT-3 to the pT + 3 position [32,37]. Curiously, substitution of pSer (pS) for pThr (pT) completely eliminates phosphopeptide binding, presumably due to a structurally conserved van der Waals interactions with the threonine y-methyl group or to entropic constraints that are unique to phosphothreonine. Tsai and co-workers found that the C-terminal FHA domain of Rad53 binds to pTyr-containing peptides in vitro [29,31] although the in vivo relevance of pTyr-dependent signaling mechanisms in budding yeast is not yet clear.

The in vivo binding partners for most FHA domain proteins are unknown, though a number of studies and clinical observations involving naturally occurring or engineered mutations or deletions within the FHA domains of key signaling molecules have verified their functional importance. Mutations that impair the ability of the N-terminal FHA domain of the yeast checkpoint kinase Rad53p to bind to phosphopeptides also result in increased sensitivity to DNA damage, whereas mutations within the FHA domain of Chk2, the human homolog of Rad53p, have been implicated in a variant form of the human cancer-prone Li-Fraumeni syndrome [38]. In addition, other mutations in FHA-domain-containing proteins including p95/Nbs1 and Chfr also appear to contribute to human tumor formation [38-41]. Chfr appears to function as a subunit of an E3 ubiquitin ligase, targeting Polo kinase for degradation to establish a cell-cycle checkpoint [42], although the role of the FHA domain in this process is not yet known.

Structures of both FHA domains from Rad53p [29,31,32], together with FHA domains from Chk2 [43] and Chfr [44], have been determined by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography (Fig. 2). The FHA domain consists of an 11-stranded P-sandwich, with a topology essentially identical to that of the MH2 domain from Smad tumor suppressor molecules [29,32].

Figure 2 FHA/phosphopeptide interactions. The FHA domain architecture as exemplified by the X-ray structure of Rad53p FHA1 in complex with a Rad9p phosphopeptide (left panel [32]). The peptide binds at one end of the P-sandwich domain in an extended conformation. The highly conserved arginine (Arg70) makes a crucial contact with the phosphate group, while a non-conserved arginine (Arg83) acts to select an Asp at the pT + 3 position (right panel, top). The phosphate group interacts with a constellation of conserved and semi-conserved FHA domain side chains (top panel), while the y-methyl group of the phosphothreonine binds in a pocket on the domain surface (right panel, bottom), likely explaining the observed preference for pThr over pSer in peptide-selection experiments. Reprinted from Yaffe and Smerdon [75], with permission.

Figure 2 FHA/phosphopeptide interactions. The FHA domain architecture as exemplified by the X-ray structure of Rad53p FHA1 in complex with a Rad9p phosphopeptide (left panel [32]). The peptide binds at one end of the P-sandwich domain in an extended conformation. The highly conserved arginine (Arg70) makes a crucial contact with the phosphate group, while a non-conserved arginine (Arg83) acts to select an Asp at the pT + 3 position (right panel, top). The phosphate group interacts with a constellation of conserved and semi-conserved FHA domain side chains (top panel), while the y-methyl group of the phosphothreonine binds in a pocket on the domain surface (right panel, bottom), likely explaining the observed preference for pThr over pSer in peptide-selection experiments. Reprinted from Yaffe and Smerdon [75], with permission.

This structural relationship, together with the remarkable similarities in phospho-dependent binding interactions of FHA and MH2 domains, suggests the existence of a super-family of FHA-like phospho-binding domains [43,45,46]. In all pThr peptide complexes, binding occurs at one end of the domain, through interactions between selected residues in the phosphopeptide and loops connecting the P3/4, P4/5, and P6/7 P-strands. Of the seven most highly conserved residues in the FHA family, three make direct interactions with the peptide (two bind directly to the pThr residue), while the remainder form the structural core or stabilize loop regions of the P-sandwich structure. Interestingly, the Chfr FHA domain structure shows a segment-swapped dimer with the C-terminal half of P7 and P8-10 exchanged between monomers. Whether or not these dimers exist in vivo or contribute to Chfr function is not known.

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