Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ

“…The dimeric status of 14-3-3 isoforms ζ, ε, γ, η and β is regulated by phosphorylation of a serineSer58 in 14-3-3ζat the dimer interface, leading to its monomerization (Figure A). , Phosphorylated, monomeric 14-3-3 is thought to sensitize cells to apoptosis, sparking interest in the development of therapeutics that stabilize it . But studying this form of 14-3-3 has been challenging due to an inability to make it: Asp/Glu phosphomimetic mutations are reported to weaken the 14-3-3 dimer, albeit poorly compared to phosphorylation, ,,, and kinase phosphorylation results in incomplete modification due to the hidden interfacial location of this residue. − Characterizations of monomeric 14-3-3 have thus historically relied on deleting the entire N-terminal dimerization interface, adding interface mutations or, very recently, a multistep strategy to purify the phosphorylated away from the unmodified form. , Using pSer and PermaPhos GCE, we expressed and then purified homogeneous 14-3-3ζ with Ser, pSer, nhpSer, as well as Glu at position S58 (Figure B), and we evaluated their oligomeric status using SEC-MALS (Figure C and Table S2). The 14-3-3ζ WT eluted as a single dimeric peak at 50 μM initial concentration (∼2-fold above physiologic concentration), as did the phosphomimetic S58E variant, consistent with previous work. , In contrast, both 14-3-3ζ pSer58 and 14-3-3ζ nhpSer58 eluted exclusively as a monomer.…”

“…10,61 Phosphorylated, monomeric 14-3-3 is thought to sensitize cells to apoptosis, sparking interest in the development of therapeutics that stabilize it. 13 But studying this form of 14-3-3 has been challenging due to an inability to make it: Asp/Glu phosphomimetic mutations are reported to weaken the 14-3-3 dimer, albeit poorly compared to phosphorylation, 17,20,28,29 and kinase phosphorylation results in incomplete modification due to the hidden interfacial location of this residue. 14−16 Characterizations of monomeric 14-3-3 have thus historically relied on deleting the entire N-terminal dimerization interface, 21 adding interface mutations or, very recently, a multistep strategy to purify the phosphorylated away from the unmodified form.…”

“…14−16 Characterizations of monomeric 14-3-3 have thus historically relied on deleting the entire N-terminal dimerization interface, 21 adding interface mutations or, very recently, a multistep strategy to purify the phosphorylated away from the unmodified form. 20,29 Using pSer and PermaPhos GCE, we expressed and then purified homogeneous 14-3-3ζ with Ser, pSer, nhpSer, as well as Glu at position S58 (Figure 7B), and we evaluated their oligomeric status using SEC-MALS (Figure 7C and Table S2). The 14-3-3ζ WT eluted as a single dimeric peak at 50 μM initial concentration (∼2-fold above physiologic concentration 20 ), as did the phosphomimetic S58E variant, consistent with previous work.…”

“…In-principle, this limitation can be overcome by substituting phospho-sites with nonhydrolyzable (i.e., stable), functional analogs or mimics (Figure ). The negatively charged amino acids aspartate and glutamate (Figure A) are trivial to incorporate into proteins, but their charge, shape and hydrogen-bonding geometries are all markedly different than those of pSer and, in the case of 14-3-3 studies, they are insufficient to promote 14-3-3/client interactions ,, and also fail to fully monomerize 14-3-3. ,,, …”

“…The negatively charged amino acids aspartate and glutamate (Figure 1A) are trivial to incorporate into proteins, but their charge, shape and hydrogen-bonding geometries are all markedly different than those of pSer and, in the case of 14-3-3 studies, they are insufficient to promote 14-3-3/client interactions 14,26,27 and also fail to fully monomerize 14-3-3. 17,20,28,29 A better structural mimic than Asp and Glu is phosphonomethyl-alanine (also called 2-amino-4-phosphonobutyrate), in which the bridging γ-oxygen of pSer is replaced with a CH 2 (Figure 1A). For simplicity, we referred to it here as "nonhydrolyzable pSer" (nhpSer).…”