The primary amino acid sequences of all 7 human 14-3-3 proteins are highly conserved (). They share about 44.4% amino acid residues that are identical in all isoforms of human 14-3-3 proteins. As shown in the phylogenetic tree in , 14-3-3σ is a more recent member of the family and evolutionarily closer to 14-3-3β and more distant from other members of the family.
Crystal structures of all seven mammalian 14-3-3 isoforms have been solved [6
]. 14-3-3σ shares many structural features with the other members of the family. All 14-3-3 molecules exist as dimers and have an overall structure that resembles a flattened horseshoe (). Each subunit of a 14-3-3 protein is composed of nine antiparallel α-helices denoted as αA to αI. Residues on αA and αB from one subunit provide interactions with the opposing residues on αC and αD from the other subunit in a dimeric complex. This arrangement of the subunits creates a central channel that has a diameter of about 15Å with a 10Å depth. Conserved salt bridges are found in 14-3-3 dimer interfaces. The salt bridge between Arg18
in one subunit and Glu91
in another has been found in all 14-3-3 isoforms while the salt bridge between Asp21
exists in all except 14-3-3ε. In addition, conserved hydrophobic interactions involving residues Leu12
are found in all 14-3-3 dimer interfaces.
Figure 2 The complex structure of 14-3-3σ and its ligand phosphopeptide. A, the complex structure of 14-3-3σ shown in perpendicular to the two-fold symmetry (PDB ID: 1YWT). The dimeric structure of 14-3-3σ is shown as ribbon while the phosphopeptide (more ...)
In contrast to the above common features shared by all human 14-3-3 family members in their dimeric structures, the structural differences between 14-3-3σ and other isoforms shed lights on its uniqueness in function and behavior. 14-3-3σ differs from other isoforms except 14-3-3γ in that it prefers to form homo-dimers whereas other isoforms can form both homo- and hetero-dimers with other members of the 14-3-3 family [13
]. Examination of the dimeric interface shows that two pairs of interactions may account for this distinctive property of 14-3-3σ (). The first one is the salt bridge between Lys9
, which occurs twice due to the 180°symmetry and exists only in 14-3-3σ. The other is the ring-ring interaction between Phe25
, which is also unique to 14-3-3σ and occurs twice [13
]. Other residues, Ser5
in 14-3-3σ are also thought to stabilize the formation of homo-dimeric complex because mutations of these three residues promoted hetero-dimerization of 14-3-3σ with other 14-3-3 isoforms [17
]. Mutations of the Phe25
residues of 14-3-3σ promoted little hetero-dimerization although they decreased the ability of 14-3-3σ to form homo-dimers [17
]. Interestingly, the combined mutations of all five residues resulted in a mutant that could no longer form homo-dimer but could form hetero-dimers with all other six 14-3-3 isoforms.
One of the important questions regarding 14-3-3σ is whether the homo-dimerization is required for its function. To answer this question, the mutant 14-3-3σ carrying mutations for all five residues as described above (Ser5
, and Gln55
) were used to determine their effect on cell proliferation [17
]. Unlike wild type 14-3-3σ, over-expression of the mutant 14-3-3σ did not inhibit the growth rate of the transfected cells [17
]. While this functional assay of cell growth rate is not the best one for 14-3-3σ, the finding appears to indicate that homo-dimerization may be required for 14-3-3σ function. Furthermore, the potential structural changes induced by these mutations are not yet known. Thus, it is not clear if the mutation-generated loss of 14-3-3σ function is due to loss of homo-dimerzation or simply due to its structural changes. It also remains to be determined if mutations of these key residues to amino acids that are different from the ones already generated would cause similar losses in dimerization properties and 14-3-3σ functions.
As discussed above, two consensus motifs containing a phosphoserine have high affinity to all 7 14-3-3 isoforms, although the presence of neither motif is required for all 14-3-3 binding partners/ligands. Based on the structure of 14-3-3σ complexed with a peptide ligand that has the RSXpSXP motif, it was thought that the major interactions between 14-3-3σ and the ligand were the same as that found in 14-3-3ζ isoform [13
]. The ligand with the RSXpSXP motif binds 14-3-3σ in a narrow basic groove composed of residues from αC, αE, αG and αI. Amino acid residues Lys49
, and Tyr130
bind and stabilize the negatively charged phosphate group of the ligand (). These conserved interactions are a consequence of strictly conserved key residues within the phosphopeptide-binding site. The selectivity of specific protein partners may involve other sites that are unique to 14-3-3 isoforms.
Interestingly, 14-3-3σ differs from other family members in the area opposite to the concave where the phosphopeptide binds. Three unique residues Met202
, and His206
in this area of 14-3-3σ are replaced by Ile, Glu, and Asp, respectively, in all other 14-3-3 isoforms (). These residues in 14-3-3σ may make more contacts with protein ligands and account for specificity and selectivity of protein partners. Indeed, mutations of these three residues of 14-3-3σ to the conserved Ile, Glu, and Asp of other family members changed the affinity of 14-3-3σ to Cdc25C [13
It is also noteworthy that a short stretch of 10–20 amino acid residues at the carboxyl terminus of 14-3-3 proteins is the most divergent region in amino acid sequences of these proteins () and all crystal structures lack this domain because no electron density could be localized in x-ray structure [6
]. This domain consists of a stretch of acidic residues and may be highly flexible. Deletion of the carboxyl terminus of 14-3-3ζ generated a protein that has higher affinity to its protein ligands [18
]. Furthermore, the binding-deficient mutant protein ligands could also bind to the mutant 14-3-3ζ lacking the carboxyl terminus. These observations suggest that the flexible carboxyl terminus of 14-3-3 proteins may play a role in regulating 14-3-3 protein binding to their ligands. However, similar studies with other 14-3-3 isoforms will be needed to confirm this conclusion.