14-3-3ζ C-terminal Stretch Changes Its Conformation upon Ligand Binding and Phosphorylation at Thr232

Abstract

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr 232 , a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the Cterminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3 C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3 at Thr 232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp 242 also revealed that phosphorylation at Thr 232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr 232 induces a more extended conformation of 14-3-3 C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.

Dysregulation of 14‐3‐3 proteins in neurodegenerative diseases with Lewy body or Alzheimer pathology

“…Phosphorylation at either site causes release of proapoptotic factors and cell death 27, 28, 29. Phosphorylation at S232 likely impacts ligand binding as the C‐terminal tail can fold back to block the binding pocket 30. We observed alterations in 14‐3‐3 phosphorylation in several PD models 31.…”

Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function

“…Time-resolved Fluorescence Measurements-Fluorescence intensity and anisotropy decays were measured on a time-correlated single photon counting apparatus, as described previously (28,41). The fluorescence decays have been acquired under the "magic angle" conditions when the measured intensity decay, I(t), is independent of the rotational diffusion of the chromophore.…”

14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4

“…Time-resolved Fluorescence Measurements-Fluorescence intensity and anisotropy decays were measured on a time-correlated single photon counting apparatus, as described previously (17,21). The fluorescence decays have been acquired under the "magic angle" conditions when the measured intensity decay, I(t), is independent of the rotational diffusion of the chromophore and provides unbiased information about lifetimes.…”

“…Tryptophan emission was collected at 355 nm through a monochromator complemented by a UG1 glass filter (Zeiss) in front of the input slit. The fluorescence decays were processed as described previously (17,21), using the singular value decomposition maximum entropy method (22).…”